A magnetic bead method low-abundance protein enrichment kit and its extraction and enzymolysis method

Abstract

This invention discloses a magnetic bead-based low-abundance protein enrichment kit and its extraction and enzymatic digestion method, relating to the field of biomedical technology. The kit includes a lysis buffer, magnetic bead washing buffer, reducing alkylation solution, two equilibration solutions, and a termination reagent. The extraction and enzymatic digestion method comprises the following steps: S1, low-abundance protein enrichment; S2, proteolytic digestion. This magnetic bead-based low-abundance protein enrichment kit and its extraction and enzymatic digestion method can rapidly and efficiently capture low-abundance proteins from bodily fluid samples, solving the problems of low digestion efficiency and frequent failures. Protein extraction is performed using an enzyme-compatible buffer, allowing the protein to be digested in the buffer, simplifying the operation. In particular, the application of multifunctional magnetic beads not only improves the enrichment efficiency of low-abundance proteins but also enhances the flexibility and throughput of analysis, opening up new prospects for proteomics research.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a magnetic bead-based low-abundance protein enrichment kit and its extraction and enzymatic digestion method. Background Technology

[0002] In the fields of biomedicine and analytical chemistry, protein extraction and analysis are crucial for understanding biological processes, disease mechanisms, and the development of novel therapies. However, traditional protein extraction methods often face significant challenges when dealing with low-abundance proteins. These traditional methods, such as centrifugation, precipitation, and conventional liquid chromatography, typically rely on the physical or chemical properties of the sample to separate and purify proteins. Because low-abundance proteins are present in extremely low concentrations (often in the picogram range or lower) in complex biological samples, traditional methods often fail to effectively identify and quantify these key biomarkers, thus limiting in-depth research into biological processes and diseases.

[0003] To address this limitation, researchers have gradually developed magnetic bead-based enrichment methods to enhance the detection of low-abundance proteins. Magnetic beads are small magnetic particles whose surfaces can be functionalized through physical or chemical modifications, allowing them to specifically bind to target proteins or other biomolecules. Compared to traditional methods, magnetic bead technology offers significant advantages. For example, the ease of handling magnetic beads makes them an ideal tool for enriching low-abundance proteins. By applying an external magnetic field, researchers can quickly separate the magnetic beads from the target protein, avoiding cumbersome centrifugation or filtration steps. This characteristic not only improves experimental efficiency but also reduces sample loss and contamination during processing. Furthermore, the high tunability of magnetic beads allows for flexible applications targeting various types of proteins or biomolecules through different functionalization strategies.

[0004] To address the above problems, this invention provides a magnetic bead-based low-abundance protein enrichment kit and its extraction and enzymatic digestion method. Summary of the Invention

[0005] The purpose of this invention is to provide a magnetic bead-based low-abundance protein enrichment kit and its extraction and enzymatic digestion method to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: On the one hand, a magnetic bead-based low-abundance protein enrichment kit is provided, comprising: lysis buffer, magnetic bead washing buffer, reducing alkylation buffer, two equilibration buffers, and a termination reagent; Lysis buffer: 1-2% sodium deoxycholate, 100 mM tris-HCl (pH = 7.6-8.5); Magnetic bead cleaning solution: 150mM KCl, 100mM tris-HCl (pH=7.6-8.5); Reduction alkylation solution: 0.33M TCEP, 0.33M CAA, 100mM tris-HCl (pH=7.6-8.5).

[0007] Furthermore, the two equilibrium solutions are specifically as follows: First equilibration solution: 1M tris-HCl (pH=7.6-8.5); The second equilibration solution: 100mM tris-HCl (pH=7.6-8.5).

[0008] Furthermore, the terminating reagent is selected from 50% formic acid (FA) or 50% trifluoroacetic acid (TFA).

[0009] On the other hand, an extraction and digestion method for a magnetic bead-based low-abundance protein enrichment kit is provided, applicable to the aforementioned magnetic bead-based low-abundance protein enrichment kit, comprising the following steps: S1. Enrichment of low-abundance proteins: The magnetic beads, samples, and magnetic bead washing solution were incubated at 37°C for 2 hours (1000 rpm, 37°C) on a constant temperature mixer, followed by washing the magnetic beads 3 times. S2, Enzymatic hydrolysis: Add 20 μL of lysis buffer and 0.6 μL of reducing alkylation solution to the cleaned magnetic beads, mix well, and react at 95°C in the dark for 5 min (1000 rpm, 95°C). Then add 1 μL of the first equilibration solution, 0.2 μg of lysyl endonuclease, and 0.6 μg of trypsin in sequence, mix well, and enzymatically hydrolyze at 37°C for 2 h (1000 rpm, 37°C).

[0010] Furthermore, step S1 specifically includes the following sub-steps: a) After the magnetic beads have returned to room temperature and been mixed, place the beads in a centrifuge tube, place the centrifuge tube on a magnetic rack and let it stand for 3 minutes until the nanoparticles are completely adsorbed, then discard the supernatant. b) Add 100 μL of magnetic bead cleaning solution to the centrifuge tube, place it on a constant temperature mixer and shake for 5 min (1000 rpm, 37°C), then place it on a magnetic rack and let it stand for 3 min until the nanoparticles are completely adsorbed. Discard the supernatant. c) Remove the sample and allow it to return to room temperature. Centrifuge at 12000g for 15 minutes at 4°C for later use. d) Resuspend the magnetic beads in the magnetic bead cleaning solution and add 40-250 μL of sample (magnetic bead cleaning solution: sample = 1:1). Place the mixture on a constant temperature mixer and incubate at 37°C with shaking for 2 hours (1000 rpm, 37°C). e) After incubation, place the centrifuge tube on a magnetic rack and let it stand for 3 minutes until the nanoparticles are completely adsorbed. Then discard the supernatant. f) Add 150 μL of magnetic bead cleaning solution to the centrifuge tube, place it on a constant temperature mixer and shake for 5 min (1000 rpm, 37 °C), then place it on a magnetic rack and let it stand for 3 min until the nanoparticles are completely adsorbed. Discard the supernatant and repeat twice.

[0011] Furthermore, step S2 specifically includes the following sub-steps: a) Add 20 μL of lysis buffer to the cleaned magnetic beads and mix thoroughly by blowing. b) Add 0.6 μL of reducing alkylation solution, mix well, and react at 95°C in the dark for 5 min (1000 rpm, 95°C). c) After heating, cool to room temperature, add 1 μL of the first equilibration solution, 0.2 μg of lysyl endonuclease and 0.6 μg of trypsin in sequence, mix well, and enzymatically hydrolyze at 37°C for 2 h (1000 rpm, 37°C). d) After the enzymatic hydrolysis is completed, add 0.4 μL of the termination reagent and mix well to terminate the enzymatic hydrolysis reaction. At this time, a precipitate will be formed. Centrifuge at 14000g for 10 min and take the supernatant. Add 100 μL of the second equilibration solution, mix well by pipetting, add 2 μL of reagent E, centrifuge and take the supernatant. Mix it with the supernatant from the first time for subsequent desalting.

[0012] This invention provides a magnetic bead-based low-abundance protein enrichment kit and its extraction and enzymatic digestion method, which has the following beneficial effects: This invention utilizes magnetic beads to capture low-abundance proteins through affinity, forming protein crowns. The proteins then undergo reductive alkylation and enzymatic cleavage on the magnetic beads, enabling rapid and efficient capture of low-abundance proteins from bodily fluid samples. This solves the problems of low enzymatic digestion efficiency and frequent failures. Protein extraction is performed using enzyme-compatible buffers, simplifying the process. In particular, the application of multifunctional magnetic beads not only improves the enrichment efficiency of low-abundance proteins but also enhances the flexibility and throughput of analysis, opening up new prospects for proteomics research. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the extraction and enzymatic digestion method of a magnetic bead-based low-abundance protein enrichment kit according to the present invention. Detailed Implementation

[0014] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0015] A magnetic bead-based low-abundance protein enrichment kit includes: lysis buffer, magnetic bead washing buffer, reducing alkylation buffer, two equilibration buffers, and a termination reagent. Lysis buffer: 1-2% sodium deoxycholate, 100 mM tris-HCl (pH = 7.6-8.5); Magnetic bead cleaning solution: 150mM KCl, 100mM tris-HCl (pH=7.6-8.5); Reduction alkylation solution: 0.33M TCEP, 0.33M CAA, 100mM tris-HCl (pH=7.6-8.5).

[0016] First equilibration solution: 1M tris-HCl (pH=7.6-8.5); The second equilibration solution: 100mM tris-HCl (pH=7.6-8.5); The terminating reagent can be 50% formic acid (FA) or 50% trifluoroacetic acid (TFA).

[0017] like Figure 1 As shown, an extraction and enzymatic digestion method for a magnetic bead-based low-abundance protein enrichment kit, applied to the aforementioned magnetic bead-based low-abundance protein enrichment kit, includes the following steps: S1, enrichment of low-abundance proteins: a) After the magnetic beads have returned to room temperature and been mixed, place the beads in a centrifuge tube, place the centrifuge tube on a magnetic rack and let it stand for 3 minutes until the nanoparticles are completely adsorbed, then discard the supernatant. b) Add 100 μL of magnetic bead cleaning solution to the centrifuge tube, place it on a constant temperature mixer and shake for 5 min (1000 rpm, 37°C), then place it on a magnetic rack and let it stand for 3 min until the nanoparticles are completely adsorbed. Discard the supernatant. c) Remove the sample and allow it to return to room temperature. Centrifuge at 12000g for 15 minutes at 4°C for later use. d) Resuspend the magnetic beads in the magnetic bead cleaning solution and add 40-250 μL of sample (magnetic bead cleaning solution: sample = 1:1). Place the mixture on a constant temperature mixer and incubate at 37°C with shaking for 2 hours (1000 rpm, 37°C). e) After incubation, place the centrifuge tube on a magnetic rack and let it stand for 3 minutes until the nanoparticles are completely adsorbed. Then discard the supernatant. f) Add 150 μL of magnetic bead cleaning solution to the centrifuge tube, place it on a constant temperature mixer and shake for 5 min (1000 rpm, 37 °C), place it on a magnetic rack and let it stand for 3 min until the nanoparticles are completely adsorbed, discard the supernatant, and repeat twice. S2, protein hydrolysis: a) Add 20 μL of lysis buffer to the cleaned magnetic beads and mix thoroughly by blowing. b) Add 0.6 μL of reducing alkylation solution, mix well, and react at 95°C in the dark for 5 min (1000 rpm, 95°C). c) After heating, cool to room temperature, add 1 μL of the first equilibration solution, 0.2 μg of lysyl endonuclease and 0.6 μg of trypsin in sequence, mix well, and enzymatically hydrolyze at 37°C for 2 h (1000 rpm, 37°C). d) After the enzymatic hydrolysis is completed, add 0.4 μL of the termination reagent and mix well to terminate the enzymatic hydrolysis reaction. At this time, a precipitate will be formed. Centrifuge at 14000g for 10 min and take the supernatant. Add 100 μL of the second equilibration solution, mix well by pipetting, add 2 μL of reagent E, centrifuge and take the supernatant. Mix it with the supernatant from the first time for subsequent desalting.

[0018] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A magnetic bead-based low-abundance protein enrichment kit, characterized in that, include: pyrolysis buffer, magnetic bead cleaning solution, reducing alkylation solution, two equilibration solutions, and termination reagent; Lysis buffer: 1-2% sodium deoxycholate, 100 mM tris-HCl; Magnetic bead cleaning solution: 150mM KCl, 100mM tris-HCl; Reductive alkylation solution: 0.33M TCEP, 0.33M CAA, 100mM tris-HCl.

2. The magnetic bead-based low-abundance protein enrichment kit according to claim 1, characterized in that, The two equilibrium solutions are as follows: First equilibrium solution: 1M tris-HCl; The second equilibrium solution is 100mM tris-HCl.

3. The magnetic bead-based low-abundance protein enrichment kit according to claim 1, characterized in that, The termination reagent is selected as 50% FA or 50% TFA.

4. The magnetic bead-based low-abundance protein enrichment kit according to claim 1, characterized in that, The pH range of the tris-HCl is 7.6 to 8.

5.

5. An extraction and enzymatic digestion method for a magnetic bead-based low-abundance protein enrichment kit, applicable to the magnetic bead-based low-abundance protein enrichment kit as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Enrichment of low-abundance proteins: The magnetic beads, samples, and magnetic bead washing solution were incubated at 37°C for 2 hours on a constant temperature mixer, followed by washing the magnetic beads 3 times. S2, Enzymatic hydrolysis: Add 20 μL of lysis buffer and 0.6 μL of reducing alkylation solution to the cleaned magnetic beads, mix well, and react at 95°C in the dark for 5 min; then add 1 μL of the first equilibration solution, 0.2 μg of lysyl endonuclease and 0.6 μg of trypsin in sequence, mix well, and hydrolyze at 37°C for 2 h.

6. The extraction and enzymatic digestion method for a magnetic bead-based low-abundance protein enrichment kit according to claim 5, characterized in that, The stirring or rotation speed of the constant temperature mixer is set to 1000 rpm.

7. The extraction and enzymatic digestion method for a magnetic bead-based low-abundance protein enrichment kit according to claim 5, characterized in that, Step S1 specifically includes the following sub-steps: a) After the magnetic beads have returned to room temperature and been mixed, place the beads in a centrifuge tube, place the centrifuge tube on a magnetic rack and let it stand for 3 minutes until the nanoparticles are completely adsorbed, then discard the supernatant. b) Add 100 μL of magnetic bead cleaning solution to the centrifuge tube, place it on a constant temperature mixer and shake for 5 min, then place it on a magnetic rack and let it stand for 3 min until the nanoparticles are completely adsorbed, and discard the supernatant. c) Remove the sample and allow it to return to room temperature. Centrifuge at 12000g for 15 minutes at 4°C for later use. d) Resuspend the magnetic beads in the magnetic bead cleaning solution, add 40-250 μL of sample, place on a constant temperature mixer, and incubate at 37°C with shaking for 2 hours; e) After incubation, place the centrifuge tube on a magnetic rack and let it stand for 3 minutes until the nanoparticles are completely adsorbed. Then discard the supernatant. f) Add 150 μL of magnetic bead cleaning solution to the centrifuge tube, shake it on a constant temperature mixer for 5 min, place it on a magnetic rack and let it stand for 3 min until the nanoparticles are completely adsorbed, discard the supernatant, and repeat twice.

8. The extraction and enzymatic digestion method for a magnetic bead-based low-abundance protein enrichment kit according to claim 6, characterized in that, In sub-step d), the amount of sample to be added is determined based on the magnetic bead cleaning solution, ensuring that the ratio of magnetic bead cleaning solution to sample is 1:

1.

9. The extraction and enzymatic digestion method for a magnetic bead-based low-abundance protein enrichment kit according to claim 5, characterized in that, Step S2 specifically includes the following sub-steps: a) Add 20 μL of lysis buffer to the cleaned magnetic beads and mix thoroughly by blowing. b) Add 0.6 μL of reducing alkylation solution, mix well, and react at 95°C in the dark for 5 min; c) After heating, cool to room temperature, add 1 μL of the first equilibration solution, 0.2 μg of lysyl endonuclease and 0.6 μg of trypsin in sequence, mix well, and enzymatically hydrolyze at 37°C for 2 h; d) After the enzymatic hydrolysis is completed, add 0.4 μL of the termination reagent and mix well to terminate the enzymatic hydrolysis reaction. At this time, a precipitate will be formed. Centrifuge at 14000g for 10 min and take the supernatant. Add 100 μL of the second equilibration solution, mix well by pipetting, add 2 μL of reagent E, centrifuge and take the supernatant. Mix it with the supernatant from the first time for subsequent desalting.

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