Hydrogen-deuterium exchange mass spectrometry detection method based on immobilized pepsin microwell plate
The stability and high-throughput problems of traditional enzymatic lysis methods are solved by immobilizing pepsin microplate, and an efficient enzyme cutting process is achieved, reducing sample loss and contamination, and improving the efficiency and reliability of hydrogen-deuterium exchange mass spectrometry experiments.
Patent Information
- Application Number
- CN202510619800.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-14
- Publication Date
- 2025-08-12
AI Technical Summary
In high-throughput proteomic research, traditional free pepsin is prone to inactivation, poor reusability, low operating stability, and enzymatic column method has problems such as low flux, high cost, high sample loss and high contamination risks, making it difficult to meet the needs of efficient and stable enzyme cutting.
Immobilized pepsin microplate is used to immobilize pepsin on the enzyme label plate and is applied to the enzyme cutting step of hydrogen-deuterium exchange mass spectrometry, avoiding the reduction of enzyme activity and sample loss, and achieving efficient loading and stability.
The stability and high throughput of the enzyme cutting process are achieved, the sample loss and contamination risks are reduced, and the efficiency and reliability of hydrogen-deuterium exchange mass spectrometry experiments are improved.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of bioengineering technology, in particular to a hydrogen-deuterium exchange mass spectrometry detection method based on an immobilized pepsin microplate. Background Art
[0002] Pepsin is a key endoprotease widely used in biomedical research, drug development, and industrial catalysis. It is particularly used in hydrogen-deuterium exchange mass spectrometry (HDX-MS) for the enzymatic hydrolysis of proteins to analyze dynamic changes in protein conformation. However, conventional free pepsin suffers from drawbacks such as rapid inactivation (pH / temperature sensitivity), poor reusability, and low operational stability, which severely restrict its application in high-throughput proteomics and industrial production.
[0003] Immobilized enzyme technology improves the stability and reusability of enzyme molecules by fixing them on the surface of the carrier (Liu Yingyu, et al. Research progress on preparation of co-immobilized nanoenzymes and their application in cascade bioreactions. Modern Food Science and Technology, 2022, 38(11): 358-366, Doi: 10.13982 / j.mfst.1673-9078.2022.11.0925.), Duan Kunfeng et al. (Duan Kunfeng, et al. Hollow fiber immobilized tyrosinase method for rapid screening of anti-melanin production active ingredients in Glehnia littoralis. Chinese Journal of Clinical Pharmacology. 2019, 35(19): 2412, Doi: 10.13699 / j.cnki.1001-6821.2019.19.057.) disclosed a method for immobilizing tyrosinase on polypropylene hollow fibers by physical adsorption, using hollow fibers made of commercially available polysulfone, polyethersulfone, polyvinylidene fluoride and polypropylene as tyrosinase carriers.
[0004] In the existing technology, enzymatic hydrolysis columns, as a solution in the current market, have improved the efficiency of enzymatic hydrolysis to a certain extent and occupied a certain share in the market. They have been widely used, especially in situations where efficient enzymatic hydrolysis is required. However, enzymatic hydrolysis columns have the following problems: 1. Low throughput: When traditional enzymatic hydrolysis methods are applied to HDX-MS experimental systems, different samples need to be replaced. Manual operations are repetitive and time-consuming, which cannot meet the needs of high-throughput experiments and limit the efficiency of large-scale proteomics research; 2. Limitations of enzymatic hydrolysis columns: Although enzymatic hydrolysis columns have improved the efficiency of enzymatic hydrolysis to a certain extent, their preparation is complex and costly, and the enzyme activity in the column may decrease over time, making it difficult to use stably for a long time; 3. Sample loss and contamination: Traditional enzymatic hydrolysis columns or solution enzymatic hydrolysis require multiple sample transfers, which increases the risk of sample loss and contamination, especially for trace samples.
[0005] In response to the above problems, the existing technology has not yet provided a solution for the enzyme cleavage step that is stable, low-cost and high-throughput. Summary of the Invention
[0006] In order to solve the above technical problems, the present invention provides a hydrogen-deuterium exchange mass spectrometry detection method based on an immobilized pepsin microplate. This method first prepares an immobilized pepsin microplate, and then applies the microplate to the enzymatic cleavage step of hydrogen-deuterium exchange mass spectrometry. The method is easy to use and has good reproducibility. It also does not require multiple transfers of protein samples, thereby reducing the risk of protein sample loss and contamination.
[0007] A hydrogen-deuterium exchange mass spectrometry detection method based on an immobilized pepsin microplate comprises the following steps:
[0008] (1) The protein to be tested is subjected to labeling reaction in hydrogen-labeled buffer and deuterium-labeled buffer respectively. After the reaction is completed, the reaction is quenched to obtain hydrogen-labeled protein solution and deuterium-labeled protein solution;
[0009] (2) dissolving pepsin with formic acid to obtain a pepsin-formic acid solution, adding the pepsin-formic acid solution to an ELISA plate, allowing it to stand, and washing it to obtain an immobilized pepsin microplate;
[0010] (3) The hydrogen-labeled protein solution and the deuterium-labeled protein solution quenched in step (1) are added to the immobilized pepsin-labeled plate obtained in step (2) for enzymatic cleavage reaction, and the peptide mixture generated by the enzymatic cleavage is separated and detected by mass spectrometry to obtain protein structure information.
[0011] In the present invention, pepsin is immobilized on an ELISA plate to achieve efficient loading of pepsin to obtain an immobilized pepsin microplate, which is then applied to the enzymatic digestion step of hydrogen-deuterium exchange mass spectrometry. Compared with the prior art enzymatic digestion column method for performing the digestion reaction, this method avoids the problem of reduced enzyme activity after repeated use, reduces the impact of residual protein samples in the enzymatic digestion column on subsequent experiments, achieves stability and high throughput of the enzymatic digestion process, and reduces sample loss and contamination risks, thereby improving the efficiency and reliability of hydrogen-deuterium exchange mass spectrometry experiments.
[0012] Preferably, in step (2), the formic acid is a 0.1% formic acid aqueous solution.
[0013] Preferably, in step (2), the pepsin activity is >2500 U / mg.
[0014] Preferably, in step (2), the concentration of pepsin in the pepsin-formic acid solution is 0.01-0.2 mg / mL.
[0015] Preferably, in step (2), the mass of pepsin in the immobilized pepsin microplate is 2 to 40 μg.
[0016] In the present invention, when the mass of pepsin is within the above range, the immobilized pepsin microplates prepared at different standing temperatures have a high coverage of the test protein after the enzymatic cleavage reaction and a small fluctuation in repeated experiments. The stability of this enzymatic cleavage method is superior to the traditional enzymatic hydrolysis column method.
[0017] Preferably, in step (2), the ELISA plate must be washed with formic acid at least three times before adding the pepsin-formic acid solution.
[0018] In the present invention, the ELISA plate needs to be cleaned at least three times with formic acid to activate the plate and simultaneously clean out impurities in the plate.
[0019] Preferably, in step (2), the ELISA plate is a 96-well ELISA plate, and the ELISA plate material is polystyrene.
[0020] In the present invention, pepsin is adsorbed on a polystyrene microplate, thereby achieving efficient loading of pepsin on the microplate and improving the stability of pepsin during the enzymatic cleavage step.
[0021] Preferably, in step (2), the standing temperature is 4-20° C., and the standing time is ≥16 h.
[0022] Preferably, in step (2), the remaining pepsin-formic acid solution is removed after standing, and the mixture is washed with a 0.1% formic acid aqueous solution.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] In the present invention, pepsin is immobilized on an ELISA plate to achieve efficient pepsin loading, resulting in an immobilized pepsin microplate. The immobilized pepsin microplate is then applied to the enzymatic digestion step of hydrogen-deuterium exchange mass spectrometry. This method reduces the impact of residual protein sample in the column on subsequent experiments during the enzymatic digestion step in the prior art, achieving stability and high throughput in the enzymatic digestion process, while reducing sample loss and contamination risks, and improving the efficiency and reliability of hydrogen-deuterium exchange mass spectrometry experiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a coverage diagram of the immobilized pepsin microplate prepared after the protein was labeled with hydrogen-labeled phosphate buffer in Examples 1 to 12 after the enzyme digestion step.
[0026] Figure 2 This is a repeated test chart of the coverage of the immobilized pepsin microplate prepared in Example 5 after the enzyme cleavage step.
[0027] Figure 3 Figure 3 shows coverage of replicate experiments after the digestion step using an enzyme column.
[0028] Figure 4 This is a comparison chart of the deuterium content of the peptides measured after microporous enzymatic digestion using the immobilized pepsin prepared in Example 5 after deuterium labeling for 1 minute, 10 minutes, and 24 hours. DETAILED DESCRIPTION
[0029] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited to the following examples.
[0030] The raw materials used in the present invention are all commercially available.
[0031] Example 1
[0032] (1) 2 μg of bovine serum albumin was subjected to deuterium labeling reaction in 20 μL of hydrogen-labeled phosphate buffer or deuterium-labeled phosphate buffer for 1 minute. After the reaction, 25 μL of quenching buffer was added and quenched at 0°C to obtain hydrogen-labeled protein solution and deuterium-labeled protein solution respectively;
[0033] (2) Dissolve 10 mg of lyophilized pepsin powder in 10 mL of 0.1% formic acid aqueous solution and dilute with 0.1% formic acid to obtain a 0.1 mg / mL pepsin-formic acid solution; wash a 96-well ELISA plate three times with 0.1% formic acid aqueous solution, then pipette 200 μL of the pepsin-formic acid solution into the ELISA plate (i.e., the amount of immobilized pepsin is 20 μg), let it stand at 4°C for 16 hours, discard the liquid in the wells, and wash with 0.1% formic acid aqueous solution to obtain an immobilized pepsin microplate;
[0034] (3) The hydrogen-labeled protein solution and the deuterium-labeled protein solution quenched in step (1) are added to the immobilized pepsin-labeled plate obtained in step (2) for 5 minutes of enzymatic digestion reaction, and the peptide mixture generated by the enzymatic digestion is separated and detected by LC-MS to analyze and obtain protein structure information.
[0035] Examples 2 to 12
[0036] The methods of Examples 2 to 12 are the same as those of Example 1, with the only difference being the preparation method of the immobilized pepsin microplate, as shown in the following table.
[0037] Table 1: Preparation differences of Examples 1 to 12
[0038]
[0039]
[0040] Sample analysis
[0041] 1. Coverage analysis after protease digestion
[0042] (1) Effect of Fixed Pepsin Amount and Standing Temperature on Coverage after Enzymatic Digestion Step Coverage tests were performed on Examples 1 to 12.
[0043] Figure 1 The coverage plots for the immobilized pepsin microplates prepared after the enzymatic digestion step, using proteins labeled with hydrogen-labeled phosphate buffer, are shown in Examples 1-12. When immobilized at 4°C, the enzymatic digestion efficiency is higher than when immobilized at 20°C, when the same amount of enzyme is immobilized. The experimental results show that the enzymatic digestion efficiency remains essentially unchanged when 16 μg or more of pepsin is immobilized.
[0044] (2) Stability test of repeated coverage experiments
[0045] The coverage test of the immobilized pepsin microplate prepared in Example 5 after enzyme digestion reaction was carried out. The results are as follows: Figure 2 shown.
[0046] 2 μg of bovine serum albumin was subjected to deuterium labeling reaction in 20 μL deuterium-labeled phosphate buffer. After the reaction, 25 μL quenching buffer was added and quenched at 0°C. The quenched bovine serum albumin was added to the immobilized pepsin enzymatic column (Immobilized Pepsin / Protease XIII Column / 2.1*33mm) for enzymatic digestion reaction. The coverage after enzymatic digestion reaction was measured. The results are as follows: Figure 3 shown.
[0047] Figure 2 This is a repeated test chart of the coverage of the immobilized pepsin microplate prepared in Example 5 after the enzyme digestion step, wherein the relative standard deviation of the coverage of 5 repeated experiments is 1.38%.
[0048] Figure 3 This is a graph showing repeated experiments of coverage after the enzymatic digestion step using an enzymatic column. The relative standard deviation of coverage after 5 repeated experiments is 5.49%. This shows that the results of enzymatic digestion using an immobilized pepsin microplate in the present application are more stable than those using an enzymatic digestion column in the prior art.
[0049] 2. Deuterium Substitution Analysis
[0050] 2 μg of bovine serum albumin was labeled in 20 μL of deuterium-labeled phosphate buffer or hydrogen-labeled phosphate buffer for 1 min, 10 min, and 24 h, respectively, and then quenched with 25 μL of quenching buffer at 0°C. Enzymatic digestion was performed using the immobilized pepsin microplate prepared in Example 5. The peptide mixture produced by enzymatic digestion was separated and detected by LC-MS. Protein structural information was analyzed and the deuterium content was calculated based on the mass data of the deuterium-labeled and unlabeled peptides.
[0051] Figure 4 Comparison of the deuterium content of peptides measured after microwell enzymatic digestion using the immobilized pepsin prepared in Example 5 after 1 minute, 10 minutes, and 24 hours of deuterium labeling. Analysis of three representative peptides extracted after the labeling and enzymatic digestion steps, and calculation of deuterium values based on mass data obtained after mass spectrometry, demonstrate that the immobilized pepsin microwell plate prepared in Example 5 can be successfully used in hydrogen-deuterium exchange mass spectrometry experiments, and that the hydrogen-deuterium exchange efficiency of bovine serum albumin increases over time within 24 hours.
[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.
Claims
1. A hydrogen-deuterium exchange mass spectrometry detection method based on an immobilized pepsin microplate, characterized in that: The following steps are involved: (1) The protein to be tested is subjected to labeling reaction in hydrogen-labeled buffer and deuterium-labeled buffer respectively. After the reaction is completed, the reaction is quenched to obtain hydrogen-labeled protein solution and deuterium-labeled protein solution; (2) dissolving pepsin with formic acid to obtain a pepsin-formic acid solution, adding the pepsin-formic acid solution to an ELISA plate, allowing it to stand, and washing it to obtain an immobilized pepsin microplate; (3) The hydrogen-labeled protein solution and the deuterium-labeled protein solution quenched in step (1) are added to the immobilized pepsin-labeled plate obtained in step (2) for enzymatic cleavage reaction, and the peptide mixture generated by the enzymatic cleavage is separated and detected by mass spectrometry to obtain protein structure information.
2. The hydrogen-deuterium exchange mass spectrometry detection method based on immobilized pepsin microplate according to claim 1, characterized in that: In step (2), the formic acid is a 0.1% formic acid aqueous solution.
3. The hydrogen-deuterium exchange mass spectrometry detection method based on immobilized pepsin microplate according to claim 1, characterized in that: In step (2), the pepsin activity is greater than 2500 U / mg.
4. The hydrogen-deuterium exchange mass spectrometry detection method based on immobilized pepsin microplate according to claim 1, characterized in that: In step (2), the concentration of pepsin in the pepsin-formic acid solution is 0.01-0.2 mg / mL.
5. The hydrogen-deuterium exchange mass spectrometry detection method based on immobilized pepsin microplate according to claim 1, characterized in that: In step (2), the mass of pepsin in the immobilized pepsin microplate is 2 to 40 μg.
6. The hydrogen-deuterium exchange mass spectrometry detection method based on immobilized pepsin microplate according to claim 1, characterized in that: In step (2), the ELISA plate must be washed at least three times with formic acid before adding the pepsin-formic acid solution.
7. The hydrogen-deuterium exchange mass spectrometry detection method based on immobilized pepsin microplate according to claim 1, characterized in that: In step (2), the ELISA plate is a 96-well ELISA plate, and the material of the ELISA plate is polystyrene.
8. The hydrogen-deuterium exchange mass spectrometry detection method based on immobilized pepsin microplate according to claim 1, characterized in that: In step (2), the standing temperature is 4-20° C., and the standing time is ≥16 h.
9. The hydrogen-deuterium exchange mass spectrometry detection method based on immobilized pepsin microplate according to claim 1, characterized in that: In step (2), the remaining pepsin-formic acid solution is removed after standing, and the mixture is washed with a 0.1% formic acid aqueous solution.