A kit based on functionalized magnetic beads and its application in the field of stem cells
The use of functionalized magnetic bead kits to enrich and identify transmembrane proteins by mass spectrometry solves the problem of difficult screening of collagen-interacting transmembrane proteins in existing technologies, and realizes efficient and simple screening and identification of transmembrane proteins, which is suitable for stem cell research and drug development.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INSTITUTE OF PROCESS ENGINEERING CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2025-12-10
- Publication Date
- 2026-07-07
AI Technical Summary
Current technologies lack methods for directly and efficiently screening and identifying collagen-interacting transmembrane proteins, hindering a deeper understanding of their downstream cellular behavior regulation mechanisms.
A functionalized magnetic bead kit was used to enrich transmembrane proteins by loading collagen-functionalized magnetic beads, and then combined with mass spectrometry identification. The kit includes mixing, washing, enzymatic digestion and mass spectrometry analysis steps to achieve efficient screening and identification of transmembrane proteins.
It enables efficient capture and screening of collagen-bound transmembrane proteins, providing a simple, stable, and highly specific tool suitable for research on the interaction between ECM and stem cell transmembrane proteins, drug target screening, and biomarker discovery.
Smart Images
Figure CN121800930B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more specifically, to a reagent kit based on functionalized magnetic beads and its application in the field of stem cells. Background Technology
[0002] Cell-extracellular matrix (ECM) interactions are a core mechanism for maintaining tissue homeostasis, regulating cell behavior, and driving disease progression. Collagen, the most abundant structural protein in the ECM, forms the "skeleton" of the ECM, providing mechanical support and regulating cellular biological behavior. Transmembrane proteins, acting as cell surface receptors, play a central role in mediating cell-extracellular matrix interactions (such as collagen). Their extracellular domains bind to ECM ligands, while their intracellular domains activate downstream signaling pathways through adaptor proteins. These interactions directly influence key biological processes such as cell adhesion, migration, proliferation, and differentiation, and are of great significance for tissue engineering, tumor metastasis, and regenerative medicine research.
[0003] Currently, there is a lack of reliable methods for directly and efficiently screening and identifying collagen-interacting transmembrane proteins. This technological gap severely restricts a deeper understanding of their downstream cellular behavior regulation mechanisms.
[0004] In view of this, the present invention is proposed. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides a product that efficiently, cost-effectively, and with high specificity enriches collagen-interacting transmembrane proteins and its application in the field of stem cells.
[0006] This invention proposes a kit based on functionalized magnetic beads, which contains functionalized magnetic beads with extracellular matrix proteins loaded on their surface. The extracellular matrix proteins are selected from collagen, preferably type I or type II collagen; the magnetic beads are preferably NHS magnetic beads.
[0007] Optionally, functionalized magnetic beads are prepared by the following method: a collagen solution is mixed with a magnetic bead suspension, coupled, washed, and separated to obtain functionalized magnetic beads.
[0008] Optionally, the kit also contains a washing solution and an enzyme digestion solution; preferably, the washing solution is a PBS solution and the enzyme digestion solution is a trypsin solution.
[0009] The present invention also proposes a method for enriching transmembrane proteins using the above-mentioned kit, comprising: binding the sample to be enriched with the above-mentioned functionalized magnetic beads to obtain the target transmembrane protein.
[0010] Optionally, the above method includes at least the following steps:
[0011] S1. Mix the sample to be enriched with functionalized magnetic beads, and load the transmembrane proteins in the sample onto the functionalized magnetic beads.
[0012] S2. Wash with cleaning solution and collect magnetic beads loaded with transmembrane proteins;
[0013] S3. Enzymatically digest the magnetic beads loaded with transmembrane proteins using an enzymatic hydrolysate, centrifuge and collect the supernatant to obtain the target transmembrane protein.
[0014] The present invention also proposes the application of the above-mentioned functionalized magnetic beads or kits in screening transmembrane proteins of stem cells.
[0015] The present invention also proposes the application of the above-mentioned kit in studying the interaction sites of stem cells and matrix or in developing drugs that target stem cells.
[0016] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:
[0017] This invention constructs a novel functionalized magnetic bead and a kit that enables the efficient capture and enrichment of collagen-bound transmembrane proteins. Combined with mass spectrometry identification, it is particularly suitable for studying the interaction between ECM and stem cell transmembrane proteins, screening drug targets, and discovering biomarkers. The kit integrates all necessary reagents, offering advantages in ease of use and speed.
[0018] This invention also constructs a method for enriching transmembrane proteins, establishing experimental methods of "magnetic bead enrichment-in-situ enzymatic digestion" and "magnetic bead enrichment-in-situ enzymatic digestion-mass spectrometry identification," enabling efficient screening and identification of collagen-bound transmembrane proteins. The method of this invention has the technical advantages of simple operation, good stability, and high specificity, providing a powerful tool for systematically studying the cell-matrix interaction mechanism mediated by transmembrane proteins, and possesses significant innovation and application potential. Attached Figure Description
[0019] Figure 1 A standard curve of collagen solution;
[0020] Figure 2 The graph shows the effect of different collagen concentrations on the coupling amount of NHS magnetic beads.
[0021] Figure 3 The graph shows the effect of different collagen volumes on the coupling amount of NHS magnetic beads.
[0022] Figure 4 A schematic diagram of the transmembrane protein enrichment and mass spectrometry identification process;
[0023] Figure 5 To extract the total ion chromatogram of transmembrane proteins;
[0024] Figure 6This is a total ion flow graph of transmembrane proteins that are not bound to collagen.
[0025] Figure 7 Total ion flux diagram of transmembrane proteins that bind to collagen;
[0026] Figure 8 This is a secondary mass spectrum of a transmembrane protein that binds to collagen. Detailed Implementation
[0027] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.
[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.
[0029] To address the shortcomings of existing technologies, this invention first constructs a novel collagen-functionalized magnetic bead system, aiming to achieve efficient capture, enrichment, and subsequent mass spectrometry identification of collagen-bound transmembrane proteins. This provides a powerful tool for systematically studying transmembrane protein-mediated cell-matrix interaction mechanisms, demonstrating significant innovation and application potential.
[0030] The functionalized magnetic beads proposed in this invention are loaded with extracellular matrix proteins on their surface, the extracellular matrix proteins being collagen. The collagen can be type I or type II collagen, and the magnetic beads are preferably NHS magnetic beads.
[0031] Specifically, this invention improves the collagen loading of NHS magnetic beads by optimizing collagen concentration and coupling volume. The process includes: preparing a collagen solution, adjusting the pH, and then adding collagen solutions of different concentrations and volumes to the NHS magnetic bead suspension. After coupling using a rotary mixer, unbound collagen is washed with PBS to obtain functionalized magnetic beads. The harvested functionalized magnetic beads are stored in PBS solution (1×PBS buffer, pH 7.2~7.4) at 4°C.
[0032] As a specific embodiment of the present invention, functionalized magnetic beads are prepared by the following method: collagen solution and magnetic bead suspension are mixed, coupled, washed and separated to obtain functionalized magnetic beads.
[0033] Specifically, the pH value of the collagen solution is 7.5 to 8.5, more preferably 7.8 to 8.2.
[0034] Specifically, in order to further increase the loading capacity of the magnetic beads, the concentration of the collagen solution can be 0.125 ~ 1.5 mg / mL, preferably 0.25 ~ 1.25 mg / mL, and more preferably 0.5 ~ 1.0 mg / mL.
[0035] Specifically, the concentration of the magnetic bead suspension is 1 to 10 mg / mL, preferably 5 to 10 mg / mL.
[0036] Specifically, the volume ratio of collagen solution to magnetic bead suspension is 1~5:1~10, preferably 2~5:1~2, and more preferably 3~5:1. The weight ratio of solute in the collagen solution to the magnetic bead suspension (i.e., the mass ratio of dry matter in the two solutions) is 0.125~1.5:10, preferably 0.5~1:10.
[0037] Specifically, the collagen solution is coupled with the magnetic beads for 1 to 3 hours, or 2 hours using a rotary mixer.
[0038] Optionally, the collagen solution is preferably prepared and used immediately. For example, it can be prepared by dissolving type II collagen in an acidic solution; the acidic solution is preferably a glacial acetic acid solution with a mass percentage of 7% to 8%.
[0039] This invention also provides a kit for enriching transmembrane proteins, which contains the aforementioned functionalized magnetic beads. Furthermore, the kit may also include a washing buffer and an enzymatic digestion buffer. The washing buffer can be a PBS solution (1×PBS buffer, pH 7.2-7.4); the enzymatic digestion buffer can be a trypsin solution (using 20 μg of sequence-grade trypsin).
[0040] This invention also proposes a method for enriching transmembrane proteins using the aforementioned functionalized magnetic beads or reagent kits. The functionalized magnetic beads bind to the sample to be enriched, yielding the target transmembrane protein. This method is simple to operate, has good stability, and high specificity, enabling efficient screening of collagen-bound transmembrane proteins.
[0041] As a specific embodiment of the present invention, the method for enriching transmembrane proteins using the above-mentioned functionalized magnetic beads or reagent kit includes at least the following steps:
[0042] S1. Mix the sample to be enriched with functionalized magnetic beads, and the functionalized magnetic beads bind to the sample to be enriched.
[0043] S2. Wash with cleaning solution to remove transmembrane proteins that are not bound to collagen, and collect magnetic beads loaded with the target protein.
[0044] S3. Enzymatic hydrolysis is performed using an enzymatic hydrolysate to dissociate the transmembrane protein from the magnetic beads. The supernatant is collected by centrifugation to obtain the enriched transmembrane protein.
[0045] In S1, the mixing is preferably rotary mixing; the mass ratio of functionalized magnetic beads to the sample to be enriched can be 1:0.5 to 5, preferably 1:0.5 to 2; the enrichment time can be 1 to 5 hours, preferably 2 to 4 hours; the enrichment temperature can be 15 to 25°C.
[0046] In S2, the washing solution is PBS solution (1×PBS buffer, pH 7.2 ~ 7.4) and vortexed for 1 ~ 4 times, preferably 2 ~ 3 times; the washing temperature can be 15 ~ 25℃.
[0047] In S3, the amount of transmembrane protein bound to the magnetic beads was determined using a BCA kit. Then, trypsin was added at a mass ratio of 20:1 to the amount of transmembrane protein to digest the collagen-functionalized magnetic beads bound to transmembrane protein in situ. The supernatant was collected by centrifugation. The digestion temperature was 36℃~38℃.
[0048] This invention also proposes a method for identifying transmembrane proteins using the aforementioned functionalized magnetic beads or the aforementioned kit. The functionalized magnetic beads bind to the sample to be enriched, yielding the target transmembrane protein. Mass spectrometry analysis is performed on the enriched transmembrane protein to obtain its peptide sequence information. Data matching of the peptide sequence information is then performed using a database to retrieve the transmembrane protein information. This method is simple to operate, stable, and highly specific, enabling efficient screening and identification of collagen-bound transmembrane proteins. It is suitable for ECM-transmembrane protein interaction studies, drug target screening, and biomarker discovery, and has significant application potential in tissue engineering and cell behavior research. It provides a powerful tool for systematically studying transmembrane protein-mediated cell-matrix interaction mechanisms.
[0049] As a specific embodiment of the present invention, the method for identifying transmembrane proteins using the above-mentioned functionalized magnetic beads or kits includes the following steps: detecting the enriched transmembrane proteins by mass spectrometry to obtain polypeptide sequence information, and performing data matching of the polypeptide sequence information through a database to retrieve information on the transmembrane proteins.
[0050] Specifically, the enriched transmembrane proteins were centrifuged, and the supernatant was analyzed by LC-MS / MS. The obtained peptides were then matched with data from the Uniprot database to retrieve collagen-interacting transmembrane proteins.
[0051] The transmembrane protein identification method proposed in this invention is simple to operate and low in cost. By systematically optimizing the collagen immobilization strategy, the capture efficiency of target proteins is significantly improved. Combined with the in-situ enzymatic digestion process, high-throughput proteomics analysis is achieved, providing a reliable technical platform for exploring the molecular mechanism of cell adhesion and developing targeted therapeutic drugs.
[0052] This invention also proposes applications for the above-mentioned kit, which can be used to screen transmembrane proteins. Based on the properties of functionalized magnetic beads, it is particularly suitable for screening stem cell transmembrane proteins, including but not limited to bone marrow mesenchymal stem cells. In addition, the above-mentioned kit is also particularly suitable for studying the interaction sites between stem cells and the matrix or developing drugs targeting stem cells. By screening stem cell transmembrane proteins that interact with collagen, transmembrane proteins related to stem cell proliferation, migration, adhesion, and differentiation are obtained. For example, key transmembrane receptors (such as integrin family members) related to stem cell proliferation and self-renewal can be identified. By identifying the screened stem cell transmembrane proteins, it is possible to understand how the collagen microenvironment maintains the undifferentiated state and continuous division capacity of stem cells through transmembrane signal transduction. The precise screening of key transmembrane proteins mediating the initial adhesion of stem cells to the collagen matrix and subsequent cytoskeleton rearrangement achieved in this invention is the first step in cell survival, proliferation, and function. In other applications, transmembrane proteins whose interactions with collagen dynamically change under different differentiation conditions (such as osteogenic and chondrogenic induction) can be screened. These proteins may be the "switch" that drives stem cell differentiation into specific lineages through microenvironmental signals. After systematically screening and identifying the collagen-interacting transmembrane proteins that are crucial for stem cell expansion, small molecules or growth factors that can activate these receptors can be added to achieve economical, efficient, and large-scale expansion of stem cells. This can then be used to identify targets, discover biomarkers, evaluate extracellular matrix materials, and assist in drug design, demonstrating broad application potential.
[0053] The present invention will be further illustrated below through specific embodiments:
[0054] Source of materials:
[0055] Bovine type II collagen was purchased from Hebei Kaolisen Biotechnology Co., Ltd.; glacial acetic acid and sodium hydroxide were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; NHS magnetic beads were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; hydrochloric acid was purchased from Shanghai Hushi Laboratory Equipment Co., Ltd.; BCA protein concentration assay kit was purchased from Beijing Lanbolide Trading Co., Ltd.; Minute TM The plasma membrane separation kit was purchased from Englishtech Biotechnology Co., Ltd.; dithiothreitol and iodoacetamide were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; sequence-grade trypsin was purchased from Promega Corporation; and acetonitrile was purchased from Thermo Fisher Scientific.
[0056] Example 1
[0057] Preparation method of functionalized magnetic beads:
[0058] 1. Weigh 6.0 mg of bovine type II collagen into a 50 mL centrifuge tube, add 6 mL of 7.5% glacial acetic acid solution to prepare a 1.0 mg / mL collagen solution, and dissolve it overnight at room temperature by magnetic stirring. The collagen solution standard curve is shown below. Figure 1 As shown. Dilute the 1.0 mg / mL collagen solution to 0.5 mg / mL, and adjust the pH to 8.0 by adding NaOH solution.
[0059] 2. Pipette the magnetic beads to resuspend them. Take 150 μL of magnetic beads (concentration of 10 mg / mL) and place them in a 2.0 mL round-bottom Eppendorf tube. Magnetic separation is used to remove the protective solution. Add 750 μL of pre-cooled 1 mM HCl to resuspend and wash the magnetic beads. Magnetic separation is used to remove the washing solution. Repeat twice.
[0060] 3. Add 150 μL of a 0.5 mg / mL collagen solution to an Eppendorf tube containing magnetic beads. Couple the tube on a rotary mixer for 2 h, then remove the coupling solution using magnetic separation to obtain functionalized magnetic beads. The harvested functionalized magnetic beads were stored in PBS solution (1×PBS buffer, pH 7.2–7.4) at 4°C.
[0061] Example 2
[0062] This embodiment presents a kit for enriching transmembrane proteins, the composition of which is shown in Table 1:
[0063] Table 1
[0064]
[0065] The instructions for using the kit are as follows:
[0066] 1. At room temperature, mix 0.2 mg of the sample to be enriched with the magnetic bead tube at a mass ratio of 1:1 and rotate to mix for 2 hours;
[0067] 2. At room temperature, the magnetic beads were separated by a magnet. The magnetic beads were then washed three times with 1.5 mL of washing solution by vortexing to remove transmembrane proteins that were not bound to collagen.
[0068] 3. At room temperature, the magnetic beads are separated by a magnet. The amount of transmembrane protein bound to the magnetic beads is determined using a BCA kit. Then, trypsin is added at a mass ratio of 20:1 to the amount of transmembrane protein. The collagen-functionalized magnetic beads bound to transmembrane proteins are digested in situ. The magnetic beads are separated by a magnet. The supernatant is obtained by centrifugation of the remaining liquid. The target protein is then obtained.
[0069] Example 3
[0070] The purpose of this embodiment is to screen the optimal concentration of the coupled collagen solution, including the following steps:
[0071] 1. Weigh 6.0 mg of bovine type II collagen into a 50 mL centrifuge tube, add 6 mL of 7.5% glacial acetic acid solution to prepare a 1.0 mg / mL collagen solution, and dissolve it overnight at room temperature by magnetic stirring. Dilute the 1.0 mg / mL collagen solution sequentially to 0.5, 0.25, and 0.125 mg / mL collagen solutions. Adjust the pH to 8.0 by adding NaOH solution.
[0072] 2. Pipette the magnetic beads to resuspend them. Take 150 μL of the magnetic beads and place them in a 2.0 mL round-bottom Eppendorf tube. Magnetic separation is used to remove the protective solution. Add 750 μL of pre-cooled 1 mM HCl to resuspend and clean the magnetic beads. Magnetic separation is used to remove the cleaning solution. Repeat twice.
[0073] 3. Take 150 μL of each of five collagen solutions with concentrations of 0, 0.125, 0.25, 0.5, and 1.0 mg / mL and add them to Ep tubes containing magnetic beads. After coupling on a rotary mixer for 2 h, remove the coupling solution by magnetic separation.
[0074] 4. The absorbance of each solution at 562 nm was measured using the BCA method to determine the coupling amount. The effect of different collagen concentrations on the coupling amount of NHS magnetic beads is shown in the graph below. Figure 2 As shown.
[0075] Example 4
[0076] The purpose of this embodiment is to determine the optimal amount of coupled collagen solution, and the difference from Example 3 is as follows:
[0077] The collagen solution concentration was 0.5 mg / mL. 150 μL, 300 μL, 450 μL, 600 μL and 750 μL of collagen solution were added to Eppendorf tubes containing magnetic beads in sequence. After coupling for 2 h, the coupling solution was removed by magnetic separation.
[0078] The absorbance of each solution at 562 nm was measured using the BCA method to determine the coupling amount. The effect of different collagen volumes on the coupling amount of NHS magnetic beads is shown in the graph below. Figure 3 As shown.
[0079] Example 5
[0080] Preparation of samples to be enriched:
[0081] Take advantage of Minute TMThe kit extracts membrane proteins from bone marrow mesenchymal stem cells and determines their concentration. The process includes: boiling the extracted membrane protein solution for 10 min to denature the proteins; adding dithiothreitol to the boiled protein solution and incubating at 60°C for 60 min to break disulfide bonds; then adding iodoacetamide to the solution and reacting at room temperature in the dark for 30 min; finally, adding 20 μg of sequence-grade trypsin, mixing thoroughly, and digesting at 37°C for 20 h; centrifuging and collecting the supernatant after digestion; and using the kit from Example 2 and following the instructions to obtain the samples to be enriched.
[0082] Example 6
[0083] A method for identifying transmembrane proteins, comprising the following steps:
[0084] The transmembrane proteins enriched in Example 5 were detected by mass spectrometry to obtain peptide sequence information. Data matching of the peptide sequences was performed using a database to retrieve transmembrane protein information. The supernatant was collected by centrifugation and analyzed by LC-MS / MS. The obtained peptides were then matched using the Uniprot database to retrieve collagen-interacting transmembrane proteins. A schematic diagram of the transmembrane protein enrichment and mass spectrometry identification process is shown below. Figure 4 As shown.
[0085] The chromatographic conditions used were as follows: Peptide CSH C18 column (1 mm × 100 mm × 1.7 μm); mobile phase A was water (containing 0.1% FA), and mobile phase B was 60% acetonitrile (containing 0.1% FA); gradient elution: 0–80 min (5%–45% B), 80–90 min (45%–90% B), 90–100 min (90% B), 100–110 min (90%–5% B); flow rate 0.1 mL / min.
[0086] The mass spectrometry conditions used were as follows: ion source spray voltage 3.5 kV; capillary temperature 320℃; sheath flow rate 19.8 mL / min; auxiliary flow rate 0.34 bar; positive ion scan mode; scan event 1 was a full scan with a scan range of m / z 400~2000, a resolution of 60000, an RF lens of 45%, a normalized AGC target of 300%, and a maximum ion implantation time of 100 ms; scan event 2 was a data dependent MS / MS with a resolution of 15000, an isolation window of m / z 1.6, a maximum ion implantation time of 200 ms, a normalized AGC target of 100%, a collision energy of 30%, and a cycle time of 2 s.
[0087] The obtained mass spectrometry data were analyzed using Protein Discoverer 2.4 software to extract the total ion chromatogram of transmembrane proteins, as shown below. Figure 5 As shown, the total ion flux diagram of transmembrane proteins not bound to collagen is as follows: Figure 6 As shown, the total ion flux diagram of transmembrane proteins bound to collagen is as follows: Figure 7 As shown, the secondary mass spectrum of transmembrane proteins bound to collagen is as follows. Figure 8 As shown, the protein database rat-transmembrane-reviewed in UniProt was used for database search to identify the types of proteins.
[0088] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A reagent kit based on functionalized magnetic beads; characterized in that, This kit contains functionalized magnetic beads, the surface of which is loaded with extracellular matrix protein, specifically type II collagen. The functionalized magnetic beads are prepared by mixing a collagen solution with a magnetic bead suspension, followed by coupling, washing, and separation to obtain the functionalized magnetic beads. The coupling time between the collagen solution and the magnetic beads is 1–3 hours, and the pH of the collagen solution is 7.8–8.
2. The concentration of the collagen solution is 0.5–1.0 mg / mL, and the weight ratio of the collagen solution to the solute in the magnetic bead suspension is 0.5:10–1:
10.
2. The reagent kit according to claim 1, characterized in that, The magnetic beads are NHS magnetic beads.
3. The reagent kit according to claim 1, characterized in that, The concentration of the magnetic bead suspension is 1 ~ 10 mg / mL.
4. The reagent kit according to claim 3, characterized in that, The concentration of the magnetic bead suspension is 5 ~ 10 mg / mL.
5. The reagent kit according to claim 1, characterized in that, The volume ratio of the collagen solution to the magnetic bead suspension is 2~5:1~2.
6. The reagent kit according to claim 5, characterized in that, The volume ratio of the collagen solution to the magnetic bead suspension is 3 to 5:
1.
7. The kit according to claim 1, characterized in that, The collagen solution is prepared by dissolving type II collagen in an acidic solution.
8. The reagent kit according to claim 7, characterized in that, The acidic solution is a glacial acetic acid solution with a mass percentage of 7-8%.
9. The reagent kit according to claim 1, characterized in that, The kit also contains a cleaning solution and an enzymatic hydrolysate.
10. The reagent kit according to claim 9, characterized in that, The washing solution is PBS solution; the enzymatic hydrolysate is pancreatic enzyme solution.
11. A method for enriching transmembrane proteins using a kit as described in any one of claims 1 to 10, characterized in that, The functionalized magnetic beads were used to bind the sample to be enriched to obtain the target transmembrane protein.
12. The method according to claim 11, characterized in that, At least the following steps are included: S1. The sample to be enriched is mixed with the functionalized magnetic beads, wherein the transmembrane proteins in the sample to be enriched are loaded onto the functionalized magnetic beads; S2. Wash with cleaning solution and collect magnetic beads loaded with transmembrane proteins; S3. The magnetic beads loaded with transmembrane protein are enzymatically hydrolyzed using an enzymatic hydrolysate, and the supernatant is collected by centrifugation to obtain the transmembrane protein.
13. The use of the kit according to any one of claims 1 to 10 in screening stem cell transmembrane proteins.
14. The kit according to any one of claims 1 to 10, used in the study of the interaction sites of stem cells and matrix or in the development of drugs targeting stem cells.