Method for carrying out in-situ enzymolysis on MauriceFlex collection liquid

Abstract

The invention belongs to the field of biology, and particularly relates to a method for carrying out in-situ enzymolysis on a MauriceFlex collecting solution. And different endonucleases are coupled through straight-chain dialdehyde substituted by iodine at 2-5 sites of C8-C30. The straight-chain dialdehyde with one end substituted by iodine is used, and the reaction activity of the aldehyde group at one end can be limited by the steric hindrance effect, so that the crosslinking degree is reduced, and the enzyme needing to be coupled is prevented from being inactivated. Besides, the used dialdehyde is a long carbon chain, so that the flexibility of the immobilized enzyme is ensured, the active site of the enzyme is convenient to fully contact with a reaction substrate in a solution, and the enzymolysis reaction is accelerated. Whether the protein is complete or not is determined through peptide fragment coverage rate analysis, and the coverage rate of straight-chain dialdehyde (2-iodine octanedialdehyde, 2, 3-diiodine octanedialdehyde and 2-iodine hexadecanedialdehyde) utilized by the method reaches 100% or is close to 100%; and the stability and the consistency of the protein can be retained to the greatest extent.

Description

Technical Field

[0001] The invention belongs to the biological field, and particularly relates to a method for in-situ enzymolysis of MauriceFlex collection fluid. Background Art

[0002] Whole-column imaged capillary isoelectric focusing (iCIEF) is a technique for separating proteins based on their isoelectric point (pI) and is widely used in the biopharmaceutical industry for the analysis of charge variants.

[0003] MauriceFlex can be used to separate and collect protein charge variant fractions. In further analysis of protein charge variant fractions, for example, when performing peptide mapping on the separated protein fractions, an endoproteinase is used to cleave the protein into peptide fragments for further analysis by mass spectrometry. This enzymatic cleavage step is usually performed after MauriceFlex fraction collection as part of subsequent analysis.

[0004] When analyzing the separated protein components, the traditional method uses free enzymes for treatment, which can achieve effective cutting for protein components with high sample amounts. However, when the amount of collected samples is small, the efficiency of free protein endonucleases is insufficient, so the efficiency of enzymatic hydrolysis needs to be further improved.

[0005] Immobilized enzyme technology is a technology that uses physical or chemical methods to fix enzymes on carriers to enable them to be reused and improve stability. Compared with free enzymes, immobilized enzymes have higher stability and reusability while maintaining efficient, specific and mild enzyme catalytic reaction characteristics. Common methods include: Chemical fixation: Use glutaraldehyde and other chemical reagents to cross-link and fix the protein endonuclease. 2 ) undergoes a cross-linking reaction to fix the enzyme on the carrier. Since the traditional glutaraldehyde cross-linking reaction conditions are relatively intense, it may cause partial inactivation of the enzyme and reduce its catalytic activity.

[0006] Physical immobilization: The enzyme is immobilized on the surface of the carrier by physical adsorption or ion exchange adsorption. This method has mild conditions, but the binding force is weak and the enzyme may fall off under high salt concentration or high temperature. Summary of the invention

[0007] In order to solve the deficiencies in the prior art, the present invention discloses a new immobilized enzyme technology, which can efficiently immobilize different protein endonucleases (such as Trypsin enzyme, Chymotrypsin enzyme or Glu-C enzyme, etc.) on a MauriceFlex 96-well plate.

[0008] In order to achieve the above object, the present invention provides the following technical solutions: The first aspect of the present invention discloses an immobilized enzyme technology, which couples different protein endonucleases via a straight-chain dialdehyde substituted with iodine at positions 2-5 of C8-C30.

[0009] Preferably, the straight-chain dialdehyde substituted with iodine at positions 2-5 of C8-C30 includes 2-iodooctanedial, 2,3-diiodooctanedial, and 2-iodohexadecanedial.

[0010] More preferably, the straight-chain dialdehyde substituted with iodine at positions 2-5 of C8-C30 is preferably 2-iodooctanedial.

[0011] The endoproteinases include Trypsin, Chymotrypsin and Glu-C.

[0012] The specific steps of the above-mentioned immobilized enzyme technology are as follows: add 100 μL of 0.05 mol / L Tris-HCl buffer solution and 80 μL of 25% of the linear dialdehyde in a 96-well plate, and cross-link for 12 hours at 25°C; wash with ultrapure water, re-add 20 μL of 0.05 mol / L Tris-HCl buffer solution and 20 μL of 0.5 mg / mL protein endonuclease, and react for 12 hours at 25°C; then wash with 0.05 mol / L Tris-HCl buffer solution, and obtain the immobilized enzyme after drying; The Tris-HCl buffer solution has a pH of 8.0 and contains 0.005 mol / L cysteine ​​and 0.002 mol / L ETA.

[0013] The second aspect of the present invention discloses the application of the above-mentioned immobilized enzyme technology in in-situ enzymolysis of MauriceFlex collected liquid. Different protein endonucleases are coupled on a MauriceFlex 96-well plate through straight-chain dialdehydes (such as 2-iodooctanedial, 2,3-diiodooctanedial, 2-iodohexadecanedial) with iodine substitution at positions 2-5 of different chain lengths (C8-C30).

[0014] Compared with the prior art, the present invention has the following technical effects: In the present invention, a straight-chain dialdehyde with iodine substitution at one end is used, and the steric hindrance effect can limit the reactivity of the aldehyde group at one end, thereby reducing the degree of cross-linking and avoiding the inactivation of the desired coupled enzyme. In addition, the dialdehyde used is a long carbon chain, which ensures the flexibility of the enzyme after fixation, facilitates the active site of the enzyme to fully contact the reaction substrate in the solution, and accelerates the enzymatic reaction.

[0015] The integrity of the protein is confirmed by peptide coverage analysis. The straight-chain dialdehydes (2-iodooctanedial, 2,3-diiodooctanedial, 2-iodohexanedialdehyde) used in the present invention have a coverage of 100% or close to 100%, which can preserve the stability and consistency of the protein to the greatest extent. DETAILED DESCRIPTION

[0016] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0017] Example 1: Synthesis of 2-iodooctanedial Synthesis route:

[0018] Synthesis method: Under nitrogen protection, suberaldehyde (2 g, 1 eq), N-iodosuccinimide (NIS, 3.5 g, 1.1 eq) and anhydrous acetonitrile (10 mL) were added to a 50 ml three-necked flask, stirred at room temperature for 10 minutes, and silver trifluoromethanesulfonate (AgOTf, 0.36 g, 0.1 eq) was added as a catalyst. The reaction was carried out at room temperature for 16 hours. 2-iodosuccinaldehyde was purified by column chromatography. 1 H NMR (400 MHz, DMSO): δ 1.19-1.39 (m, 4H), 1.52 (tt, 2H), 1.94 (q, 2H), 2.51 (td, 2H), 3.90 (td,1H), 9.65 (t, 1H), 9.92 (d, 1H).

[0019] Example 2: Synthesis of 2,3-diiodooctanedial Synthesis route:

[0020] Synthesis method: Under nitrogen protection, suberaldehyde (2 g, 1 eq), N-iodosuccinimide (NIS, 3.5 g, 1.1 eq) and anhydrous acetonitrile (10 mL) were added to a 50 ml three-necked flask and stirred at 40 °C for 10 minutes. Indium trifluoromethanesulfonate (In(OTf) 3 , 0.8 g, 0.1 eq) as catalyst. The reaction was carried out at 40 °C for 24 hours. 2,3-diiodooctanedial was obtained by column chromatography purification. 1 H NMR (400 MHz, DMSO): δ 0.79 (tt, 2H), 1.40 (q, 2H), 1.54 (tt, 2H), 2.52 (td, 2H), 3.34 (td,1H), 4.26 (dd, 1H), 9.65 (t, 1H), 9.96 (d,1H).

[0021] Example 3: Synthesis of 2-iodohexadecanedialdehyde Synthesis route:

[0022] Synthesis method: Under nitrogen protection, add some diatomaceous earth to a 50 ml three-necked flask. Then add hexadecanediol (2 g, 1 eq), pyridinium dichromate (PDC, 3.5 g, 1.5 eq) and dichloromethane (20 mL), stir at 30 °C for 20 hours. Purify by column chromatography to obtain hexadecanedial.

[0023] Under nitrogen protection, add hexadecanedial (1 g, 1 eq), N-iodosuccinimide (NIS, 0.96 g, 1.1 eq) and anhydrous acetonitrile (10 mL) into a 50 ml three-necked flask, stir at room temperature for 10 minutes, add silver trifluoromethanesulfonate (AgOTf, 0.1 g, 0.1 eq) as a catalyst, react at room temperature for 30 hours, and purify by column chromatography to obtain 2-iodohexadecanedial. 1 H NMR (400 MHz, DMSO): δ 1.16-1.36 (m, 20H), 1.51 (tt, 2H), 1.94 (dt, 2H), 2.51(td, 2H), 3.91 (td, 1H), 9.64 (t, 1H), 9.93 (d, 1H).

[0024] Example 4: Enzyme immobilization and enzymatic protein hydrolysis Enzyme immobilization: Add 100 μL of 0.05 mol / L Tris-HCl buffer solution (pH 8.0, containing 0.005 mol / L cysteine ​​and 0.002 mol / L EDTA) and 80 μL of 25% linear dialdehyde to a 96-well plate, and cross-link for 12 hours at 25 °C. Wash with ultrapure water. Add 20 μL of 0.05 mol / L Tris-HCl buffer solution (pH 8.0, containing 0.005 mol / L cysteine ​​and 0.002 mol / L EDTA) and 20 μL of Trypsin enzyme (0.5 mg / mL) again, and react at 25 °C for 12 hours. Wash with 0.05 mol / L Tris-HCl buffer solution (pH 8.0, containing 0.005 mol / L cysteine ​​and 0.002 mol / EDTA), and dry to obtain the immobilized enzyme.

[0025] Enzymatic protein: Protein samples were collected using a 96-well plate with immobilized Trypsin enzyme, and 30 μL of enzymatic digestion buffer was added to the corresponding wells and mixed. Microwave-assisted enzymatic digestion was performed for 1 min. After the reaction, the protein sample coverage was detected by LC-MS / MS.

[0026] Coverage results: The data obtained by LC-MS / MS were searched in the database, and the results are shown in the table below.

[0027] Table 1 Serial number Straight chain dialdehyde Coverage 1 2-Iodooctanedial 100% 2 2,3-Diiodooctanedial 95% 3 2-Iodohexadecanedial 99% 4 Glutaraldehyde 30% Example 5: Free Trypsin enzyme Enzymatic protein: Use a common 96-well plate to collect protein samples, add 30 μL of enzymatic digestion buffer and 2 μL of Trypsin enzyme solution (0.5 mg / mL) to the corresponding wells, and mix well. Microwave-assisted enzymatic digestion for 1 min. After the reaction, the protein sample coverage was detected by LC-MS / MS.

[0028] Coverage results: The data obtained by LC-MS / MS was searched in the database, and the coverage result was 45%.

[0029] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An immobilized enzyme technology, characterized in that: Different protein endoenzymes are coupled via straight-chain dialdehydes substituted with iodine at positions 2-5 of C8-C30.

2. An immobilized enzyme technology according to claim 1, characterized in that: The straight-chain dialdehyde substituted with iodine at positions 2-5 of C8-C30 includes 2-iodooctanedial, 2,3-diiodooctanedial, and 2-iodohexadecanedial.

3. An immobilized enzyme technology according to claim 2, characterized in that: The straight-chain dialdehyde substituted with iodine at positions 2-5 of C8-C30 is preferably 2-iodooctanedial.

4. The immobilized enzyme technology according to claim 1, characterized in that: The protein endoenzyme includes Trypsin enzyme, Chymotrypsin enzyme and Glu-C enzyme.

5. The immobilized enzyme technology according to claim 1, characterized in that: The specific steps are as follows: add 100 μL of 0.05 mol / L Tris-HCl buffer solution and 80 μL of 25% of the linear dialdehyde in a 96-well plate, and perform cross-linking reaction at 25°C for 12 hours; Wash with ultrapure water, add 20 μL of 0.05 mol / L Tris-HCl buffer solution and 20 μL of 0.5 mg / mL protein endonuclease, react at 25 °C for 12 hours; then wash with 0.05 mol / L Tris-HCl buffer solution, and dry to obtain the immobilized enzyme; The Tris-HCl buffer solution has a pH of 8.0 and contains 0.005 mol / L cysteine ​​and 0.002 mol / L ETA.

6. Use of the immobilized enzyme technology according to any one of claims 1 to 5 in in situ enzymatic hydrolysis of MauriceFlex collected fluid.

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