A method for in-situ enzymatic hydrolysis of MauriceFlex collection fluid

Abstract

The present invention belongs to the field of biology, and specifically relates to a method for in-situ enzymatic hydrolysis of MauriceFlex collection solution. A straight-chain dialdehyde with iodine substitution at the 2-5 positions of C8-C30 is coupled with different endoproteinases. By using a straight-chain dialdehyde with iodine substitution at one end, the steric hindrance effect can limit the reactivity of one aldehyde group, thereby reducing the degree of crosslinking and avoiding the inactivation of the enzyme to be coupled. In addition, the dialdehyde used has a long carbon chain, ensuring the flexibility after enzyme immobilization, facilitating the full contact between the active site of the enzyme and the reaction substrate in the solution, and accelerating the enzymatic hydrolysis reaction. Whether the protein is intact is confirmed by peptide segment coverage analysis. The straight-chain dialdehydes (2-iodooctanedial, 2,3-diiodooctanedial, 2-iodohexadecanedial) utilized in the present invention have a coverage rate of 100% or close to 100%; the stability and consistency of the protein can be maximally retained.

Description

Technical Field

[0001] The present invention belongs to the field of biology, and particularly relates to a method for in-situ enzymatic hydrolysis of MauriceFlex collection solution. Background Art

[0002] Imaged capillary isoelectric focusing (iCIEF) is a technique for separating proteins based on their isoelectric points (pI), and is widely used in the biopharmaceutical industry for the analysis of charge variants.

[0003] MauriceFlex can be used for the separation and collection of protein charge variant components. In the further analysis of protein charge variant components, for example, when performing peptide mapping analysis on the separated protein components, endoproteases are used to cleave the proteins into polypeptide fragments for further analysis by mass spectrometry. This enzymatic cleavage step is usually carried out after the component collection of MauriceFlex as part of the subsequent analysis.

[0004] When analyzing the separated protein components, in traditional methods, free enzymes are used for treatment, which can achieve effective cleavage for protein components with a high sample amount. However, when the collected sample amount is small, the continuous enzymatic hydrolysis efficiency of free endoproteases is insufficient, so it is necessary to further improve the enzymatic hydrolysis efficiency.

[0005] The immobilized enzyme technology is a technology that immobilizes enzymes on carriers through physical or chemical methods, enabling them to be reused and improving stability; compared with free enzymes, immobilized enzymes not only maintain the characteristics of highly efficient, specific, and mild enzymatic catalytic reactions, but also have higher stability and reusability. Common methods include:

[0006] Chemical immobilization: Crosslinking and immobilizing endoproteases using chemical reagents such as glutaraldehyde. It can crosslink and immobilize the enzyme on the carrier by reacting with the amino groups (-NH2) on the enzyme molecule. Since the traditional glutaraldehyde crosslinking reaction conditions are relatively harsh, it may cause partial inactivation of the enzyme and reduce its catalytic activity.

[0007] Physical immobilization: Immobilizing the enzyme on the surface of the carrier through physical adsorption or ion exchange adsorption. This method has mild conditions, but the binding force is weak, and the enzyme may fall off at high salt concentrations or high temperatures. Summary of the Invention

[0008] To address the deficiencies in the prior art, the present invention discloses a new immobilized enzyme technology that can efficiently immobilize different endoproteinases (such as Trypsin, Chymotrypsin, or Glu-C enzyme, etc.) on a 96-well plate of MauriceFlex.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The first aspect of the present invention discloses an immobilized enzyme technology that couples different endoproteinases through a linear dialdehyde with iodine substitution at the 2-5 positions of C8-C30.

[0011] Preferably, the linear dialdehyde with iodine substitution at the 2-5 positions of C8-C30 includes 2-iodooctanedial, 2,3-diiodooctanedial, 2-iodohexadecanedial.

[0012] More preferably, the linear dialdehyde with iodine substitution at the 2-5 positions of C8-C30 is preferably 2-iodooctanedial.

[0013] Wherein the endoproteinases include Trypsin enzyme, Chymotrypsin enzyme, and Glu-C enzyme.

[0014] The specific steps of the above immobilized enzyme technology are as follows: Add 100 μL of 0.05 mol / L Tris-HCl buffer solution, 80 μL of 25% of the linear dialdehyde into a 96-well plate, and carry out a cross-linking reaction at 25 °C for 12 hours; wash with ultrapure water, then add 20 μL of 0.05 mol / L Tris-HCl buffer solution and 20 μL of endoproteinase with a concentration of 0.5 mg / mL, and react at 25 °C for 12 hours; wash again with 0.05 mol / L Tris-HCl buffer solution, and obtain the immobilized enzyme after drying;

[0015] The pH of the Tris-HCl buffer solution is 8.0, and it contains 0.005 mol / L cysteine and 0.002 mol / L EDTA.

[0016] The second aspect of the present invention discloses the application of the above immobilized enzyme technology in in-situ enzymatic hydrolysis of the MauriceFlex collection solution. Different endoproteinases are coupled on a 96-well plate of MauriceFlex through linear dialdehydes with iodine substitution at the 2-5 positions of different chain lengths (C8-C30) (such as 2-iodooctanedial, 2,3-diiodooctanedial, 2-iodohexadecanedial).

[0017] Compared with the prior art, the present invention has the following technical effects:

[0018] In the present invention, a linear dialdehyde with iodine substitution at one end is used. The steric hindrance effect can limit the reactivity of one aldehyde group, thereby reducing the crosslinking degree and avoiding the inactivation of the enzyme to be coupled. In addition, the dialdehyde used has a long carbon chain, ensuring the flexibility after enzyme immobilization, facilitating the full contact between the active site of the enzyme and the reaction substrate in the solution, and accelerating the enzymatic hydrolysis reaction.

[0019] To confirm the integrity of the protein through peptide coverage analysis, the linear dialdehydes (2-iodooctanedial, 2,3-diiodooctanedial, 2-iodohexadecanedial) utilized in the present invention have a coverage rate reaching 100% or approaching 100%; the stability and consistency of the protein can be maximally retained. Detailed implementation mode

[0020] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods in the following examples are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0021] Example 1: Synthesis of 2-iodooctanedial

[0022] Synthesis route:

[0023]

[0024] Synthesis method:

[0025] Under nitrogen protection, add octanedial (2 g, 1 eq), N-iodosuccinimide (NIS, 3.5 g, 1.1 eq) and anhydrous acetonitrile (10 mL) into a 50 ml three-necked flask, stir at room temperature for 10 minutes, and add silver trifluoromethanesulfonate (AgOTf, 0.36 g, 0.1 eq) for catalysis. React at room temperature for 16 hours. Purify by column chromatography to obtain 2-iodooctanedial. 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).

[0026] Example 2: Synthesis of 2,3-diiodooctanedial

[0027] Synthesis route:

[0028]

[0029] Synthesis method:

[0030] Under nitrogen protection, add octanedial (2 g, 1 eq), N-iodosuccinimide (NIS, 3.5 g, 1.1 eq) and anhydrous acetonitrile (10 mL) into a 50 mL three-necked flask, stir at 40 °C for 10 minutes, and add indium(III) trifluoromethanesulfonate (In(OTf)3, 0.8 g, 0.1 eq) as a catalyst. React at 40 °C for 24 hours. Purify by column chromatography to obtain 2,3-diiodooctanedial. 1 1H 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).

[0031] Example 3: Synthesis of 2-iodohexadecanedial

[0032] Synthesis route:

[0033]

[0034] Synthesis method:

[0035] Under nitrogen protection, add some diatomaceous earth into 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), and stir at 30 °C for 20 hours. Purify by column chromatography to obtain hexadecanedial.

[0036] 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, and add silver trifluoromethanesulfonate (AgOTf, 0.1 g, 0.1 eq) as a catalyst. React at room temperature for 30 hours. Purify by column chromatography to obtain 2-iodohexadecanedial. 1 1H 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).

[0037] Example 4: Enzyme immobilization and enzymatic hydrolysis of protein

[0038] 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) into a 96-well plate, then add 80 μL of 25% linear dialdehyde, and perform a cross-linking reaction at 25 °C for 12 hours. Wash with ultrapure water. Re-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), and react at 25 °C for 12 hours. Wash again with 0.05 mol / L Tris-HCl buffer solution (pH 8.0, containing 0.005 mol / L cysteine and 0.002 mol / L EDTA), and obtain the immobilized enzyme after drying.

[0039] Protein Enzymolysis:

[0040] Use the 96-well plate immobilized with Trypsin enzyme to collect protein samples, add 30 μL of enzymolysis buffer into the corresponding wells, and mix well. Perform microwave-assisted enzymolysis for 1 min. After the reaction, detect the protein sample coverage rate by LC-MS / MS.

[0041] Coverage Rate Results: Search the data obtained by LC-MS / MS in the database, and the results are shown in the following table.

[0042] Table 1

[0043] Serial number Linear dialdehyde Coverage rate 1 2-Iodooctanedial 100% 2 2,3-Diiodooctanedial 95% 3 2-Iodohexadecanedial 99% 4 Glutaraldehyde 30%

[0044] Example 5: Free Trypsin Enzyme

[0045] Protein Enzymolysis:

[0046] Use a common 96-well plate to collect protein samples, add 30 μL of enzymolysis buffer and 2 μL of Trypsin enzyme solution (0.5 mg / mL) into the corresponding wells, and mix well. Perform microwave-assisted enzymolysis for 1 min. After the reaction, detect the protein sample coverage rate by LC-MS / MS.

[0047] Coverage Rate Results: Search the data obtained by LC-MS / MS in the database, and the coverage rate result is 45%.

[0048] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

Claims

1. An immobilized enzyme method, characterized in that, Couple different endoproteinases with 2-iodooctanedial, 2,3-diiodooctanedial, and 2-iodohexadecanedial.

2. The immobilized enzyme method according to claim 1, wherein, Couple different endoproteinases with 2-iodooctanedial.

3. The immobilized enzyme method according to claim 1, characterized in that, The endoproteinases include Trypsin, Chymotrypsin, and Glu-C.

4. A method for immobilizing an enzyme according to claim 1, wherein, The specific steps are as follows: Add 100 μL of 0.05 mol / L Tris-HCl buffer solution and 80 μL of 25% linear dialdehyde into a 96-well plate, and conduct a cross-linking reaction at 25 °C for 12 hours. Wash with ultrapure water, re-add 20 μL of 0.05 mol / L Tris-HCl buffer solution and 20 μL of endoproteinase with a concentration of 0.5 mg / mL, and react at 25 °C for 12 hours; then wash with 0.05 mol / L Tris-HCl buffer solution, and obtain the immobilized enzyme after drying. The pH of the Tris-HCl buffer solution is 8.0, and it contains 0.005 mol / L cysteine and 0.002 mol / L EDTA.

5. Application of the immobilized enzyme prepared by the immobilized enzyme method according to any one of claims 1-4 in in-situ enzymatic hydrolysis of the MauriceFlex collection solution.

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