Method for separating and purifying cell surface protein and application thereof

Abstract

The application provides a method for separating and purifying cell surface proteins and application thereof. The method comprises the following steps: culturing cells to be separated, adding an azide solution to the cells for incubation, then adding a Tris-HCl buffer to terminate the reaction, so as to label the cell surface proteins with azides; then adding a TNTE buffer and a protease inhibitor to lyse the cells, centrifuging to obtain cell lysates labeled with azides; incubating the cell lysates labeled with azides with phosphine-biotin-streptavidin magnetic beads, so as to enrich the cell surface proteins labeled with azides, and obtain magnetic beads containing the cell surface proteins. The method can effectively separate the cell surface proteins, has low background, high efficiency and good repeatability, and can be applied to quantitative analysis of cell surface proteomes.

Description

Technical Field

[0001] This invention belongs to the field of protein purification technology, and relates to a method for isolating and purifying cell surface proteins and its application. Background Technology

[0002] Cell surface proteins play a crucial role in cell-to-cell recognition and cell-microenvironment interactions. Cell surface biotinylation has long been one of the most commonly used methods for surface proteomics analysis. Currently, the method of directly capturing biotinylated cell surface proteins using streptavidin-based magnetic beads is widely applied. However, this traditional biotinylation method is affected by endogenous biotin-associated proteins (BATs). Surface biotinylation has been widely used to analyze cell membrane-related proteomics. However, the workflow can be interfered with by cytoplasmic biotin-associated proteins, which compete with streptavidin binding during purification, interfering with the accuracy and efficiency of quantitative analysis. Summary of the Invention

[0003] In view of the deficiencies in the existing technology, one object of the present invention is to provide a method for isolating and purifying cell surface proteins. Another object of the present invention is to provide the application of this method for isolating and purifying cell surface proteins in the quantitative analysis of cell surface proteome.

[0004] The present invention provides a method for isolating and purifying cell surface proteins using a bioorthogonal conjugation-assisted purification (BCAP) method. This method utilizes the Staudinger chemoselective ligation technique to label and isolate cell surface-related proteins, thereby minimizing interference from endogenous biotin-associated proteins. In the BCAP workflow, exposed cell surface proteins are first labeled with NHS-PEG4-Azide. Then, after terminating the reaction with 100 nM Tris-HCl, TNTE is added to lyse the cells and obtain lysates. Simultaneously, Phosphine-PEG3-Biotin and streptavidin-coated magnetic beads are incubated together, ensuring complete coating of all streptavidin sites on the beads with Phosphine-PEG3-Biotin. The coated phosphine-biotin-streptavidin magnetic beads are then incubated with the cell lysates, and the NHS-PEG4-Azide-labeled cell surface proteins are enriched through a bioorthogonal reaction between azide and phosphine. The method of the present invention effectively isolates cell surface proteins and exhibits high reproducibility.

[0005] Specifically, on the one hand, the present invention provides a method for isolating and purifying cell surface proteins, which includes the following steps:

[0006] Cells to be isolated were cultured, and an azide solution was added to the cells for incubation. The reaction was then terminated by adding Tris-HCl buffer to label the cell surface proteins with azides.

[0007] Next, TNTE buffer and protease inhibitor were added to lyse the cells, and after centrifugation, azide-labeled cell lysates were obtained.

[0008] Azide-labeled cell lysates were incubated with phospho-biotin-streptavidin magnetic beads to enrich azide-labeled cell surface proteins, resulting in magnetic beads containing cell surface proteins.

[0009] The phosphine-biotin-streptavidin magnetic beads are obtained by incubating and washing phosphine-polyethylene glycol-biotin with streptavidin magnetic beads.

[0010] According to a specific embodiment of the present invention, the method for separating and purifying cell surface proteins of the present invention further includes: adding magnetic beads containing cell surface proteins to an elution buffer for incubation and elution to elute the cell surface proteins from the magnetic beads.

[0011] According to a specific embodiment of the present invention, the method for separating and purifying cell surface proteins of the present invention further includes: enzymatically digesting the proteins separated and enriched from cells to obtain peptides of cell surface proteins.

[0012] In the above-described separation and purification method, preferably, the cells to be separated include human non-small cell lung cancer A549 cells and / or mouse embryonic fibroblasts; but not limited thereto.

[0013] In the above separation and purification method, preferably, the azide solution is a 10 mM azide solution prepared with PBS buffer at pH 8.0.

[0014] In the above separation and purification method, preferably, the azide includes succinimide acrylate-polyethylene glycol-azide (NHS-PEG4-Azide); but it is not limited to this.

[0015] In the above separation and purification method, preferably, the concentration of the Tris-HCl buffer is 100 mM and the pH value is 7.4.

[0016] In the above separation and purification method, preferably, the reaction is terminated by adding Tris-HCl buffer for 5 minutes.

[0017] In the above separation and purification method, preferably, the temperature for incubating the cells with azide solution is 4°C and the incubation time is 1 hour.

[0018] In the above separation and purification method, preferably, the TNTE buffer contains 50 mM Tris-HCl with a pH of 7.4, 150 mM NaCl, 1% Triton-X100 and 1 mM EDTA.

[0019] In the above separation and purification methods, preferably, cell lysis is performed in an ice bath for 30 minutes.

[0020] In the above separation and purification method, preferably, the phosphine-polyethylene glycol-biotin solution is a 0.05 mM phosphine-polyethylene glycol-biotin solution prepared with PBS buffer at pH 7.4. In the above separation and purification method, preferably, the phosphine-polyethylene glycol-biotin is incubated with streptavidin magnetic beads at room temperature for 1 hour.

[0021] In the above separation and purification method, preferably, the temperature for incubating the azide-labeled cell lysate with phospho-biotin-streptavidin magnetic beads is 37°C and the incubation time is 4 hours.

[0022] In the above separation and purification method, preferably, the washing after incubation is performed using PBS buffer with a pH of 7.4.

[0023] According to a specific embodiment of the present invention, the process of adding magnetic beads containing cell surface proteins to elution buffer for incubation and elution includes: adding elution buffer I to magnetic beads containing cell surface proteins for incubation for 1 hour, then adding elution buffer II to wash the magnetic beads and collecting the supernatant.

[0024] In the above separation and purification method, preferably, the elution buffer I includes: 2M urea, 50mM Tris-HCl pH 8.0, 1mM DTT, and 10μg / mL sequencing-grade trypsin.

[0025] In the above separation and purification method, preferably, the eluent II comprises: 2M urea, 50mM Tris-HCl pH 8.0, and 5mM iodoacetamide.

[0026] On the other hand, the present invention also provides a phosphine-biotin-streptavidin magnetic bead for the separation and purification of said cell surface proteins, which is obtained by incubating and washing phosphine-polyethylene glycol-biotin with streptavidin magnetic beads.

[0027] On the other hand, the present invention also provides the use of azides as markers in labeling cell surface proteins. Preferably, the azides comprise succinimide acrylate-polyethylene glycol-azide.

[0028] On the other hand, the present invention also provides the application of the above-mentioned method for isolating and purifying cell surface proteins in the quantitative analysis of cell surface proteome.

[0029] The beneficial effects of this invention are as follows: This invention employs a biological orthogonal linkage-assisted purification (BCAP) method to separate and purify cell surface proteins, which can effectively separate cell surface proteins with low background, high efficiency, and good reproducibility. In addition, the cell surface protein separation and purification method of this invention can be applied to the comparative analysis of cell surface proteins under different conditions, enabling efficient and reproducible cell surface proteomics analysis. When this method is applied to compare the differences in surface membrane proteins of proliferating and senescent mouse fibroblasts (MEFs), this invention also discovered that EHD2 has enhanced cell membrane localization in senescent MEFs. Attached Figure Description

[0030] Figure 1 This diagram compares the workflow of separating cell surface proteins using the Bio-Orthogonal Linkage-Assisted Purification (BCAP) method of this invention and the conventional sulfonyl-NHS-SS-Biotin method (the BCAP method of this invention aims to minimize the competitive binding of endogenous biotin-related proteins).

[0031] Figure 2A To detect A549 cells with or without the NHS-PEG4-Azide label using DBCO-Cy5 and DAPI, scale bar 20 μm.

[0032] Figure 2B Western blot analysis of proteins prepared for cell surface (where A549 cells were treated with a specified labeling procedure before cell lysis; whole cell lysates were incubated with streptavidin beads or phosphobiotin-streptavidin beads; and isolated proteins were biotinylated using HRP-streptavidin).

[0033] Figure 2C To produce Western blot images of proteins for BCAP in the absence or presence of 0.25% trypsin / EDTA treatment (Biotin blot of isolated proteins using HRP-streptavidin).

[0034] Figure 3A A Venn diagram showing the number of surface-associated proteins identified by two biological replicates using a single method.

[0035] Figure 3B The Venn diagram shows the overlap of proteins identified by two methods after combining data from two replicates.

[0036] Figure 3C This is a graph showing the top five enrichment categories for GO enrichment analysis of cellular compartments of proteins identified by the two methods.

[0037] Figure 3D A graph showing the number of proteins in a specified category for GO enrichment analysis of cellular compartments of proteins identified by the two methods.

[0038] Figure 3E Comparative plot of quantitative reproducibility of proteins isolated by the Sulfo-NHS-SS-Biotin method (left) and the BCAP method (right) workflow (histograms of log10 transformation abundance of isolated proteins from two biological replicates are distributed along the x and y axes, respectively).

[0039] Figure 4A A comparative diagram of surface-associated proteins isolated from proliferating and senescent MEFs using the BCAP method (which combines the results of two biological replicates).

[0040] Figure 4B A graph showing GO enrichment analysis of cellular compartments of proteins identified from proliferating and senescent MEFs using the BCAP method.

[0041] Figure 4C Volcano plot for differential surface proteome analysis between proliferating and senescent MEFs (vertical and horizontal dashed lines delineate absolute fold changes ≥2 and adjusted p-values ​​≤0.05, respectively).

[0042] Figure 5A Western blot images of total cell lysates and proteins enriched by the BCAP method in proliferating and senescent MEFs, detected with anti-EHD2 antibody.

[0043] Figure 5B Percentage of cells with positive immunomarkers for EHD2 in proliferating (P) and senescent (S) MEFs in the absence or presence of Triton X-100-mediated permeabilization (Results are described as mean ± SEM from 3 replicates, each quantified >50 cells).

[0044] Figure 5C A representative immunomarker of EHD2 in proliferative (P) and senescent (S) MEFs in the absence or presence of Triton X-100-mediated permeabilization.

[0045] Figure 6A The plot shows the culture and reproduction time of MEF and the corresponding population multiplication level (PDL).

[0046] Figure 6B Representative plot of senescence-associated β-galactosidase (SA-β-gal) staining for proliferating and senescent MEFs (the percentage of positive cells was quantified in the right plot (results described as mean ± SEM, N = 3)). Detailed Implementation

[0047] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0048] Unless otherwise specified, all original reagents and materials used in the examples were commercially available. Methods not detailed in the examples should be performed according to standard operating conditions in the relevant field or the operating conditions recommended in the instrument manufacturer's instructions.

[0049] Example 1:

[0050] 1. Cell culture:

[0051] Human non-small cell lung cancer (NSCLC) A549 (ATCC CCL-185) cells were cultured in DMEM medium containing 10% FBS, 2 mM L-glutamine and 100 μg / ml penicillin-streptomycin (Thermo Fisher).

[0052] 2. Isolation and culture of mouse embryonic fibroblasts (MEF):

[0053] This study used C57BL / 6J mice cultured in 12-hour light and 12-hour dark cycles. All mouse experiments were conducted in accordance with protocols approved by the Institutional Animal Research Ethics Subcommittee of City University of Hong Kong and the Department of Health of the Hong Kong Special Administrative Region Government.

[0054] To obtain E13.5 mouse embryos, male and female mice aged 8 to 15 weeks were mated. Pregnant female E13.5 mice were anesthetized by inhalation with 4% isoflurane, and embryos were then removed from the ovaries. Immediately after embryo removal, the pregnant mice were euthanized by decapitation; the embryonic tissue was then minced with a blade in ice-cold PBS (pH 7.4) buffer. The tissue fragments were then incubated in 0.25% trypsin at 37°C for 15 minutes, followed by centrifugation at 1500 rpm for 5 minutes at 4°C. 2 mL of DMEM medium (containing 10% FBS and 1% penicillin-streptomycin, both from Thermo Fisher) was added to the tissue pellet and stirred. Finally, the cell solution was filtered through a 75 μm filter and centrifuged at 75 cm⁻¹. 2 Cells were cultured in culture flasks. To determine the cumulative population multiplication rate (PDL), initial seeding cell counts, final viable cell counts, and the time to 90% confluence were recorded. PDL was calculated using the following formula:

[0055] PDL = 3.32(logXf - logXi) + S

[0056] In the formula, S represents the PDL at the start of culture, Xf represents the final surviving cell count, and Xi is the initial seeded cell count.

[0057] 3. Cell surface protein labeling and separation (main process as follows) Figure 1 As shown):

[0058] (1) 5×10 6 A549 cells or mouse embryonic fibroblasts (MEFs) were washed three times with ice-cold PSB (pH 7.4) buffer.

[0059] (2) Add succinimide acrylate-polyethylene glycol-azide (NHS-PEG4-Azide, ThermoScientific, product #26130) to PBS (pH 7.4) buffer to prepare a 10 mM NHS-PEG4-Azide solution.

[0060] (3) At 4℃, A549 cells or mouse embryonic fibroblasts (MEF) were mixed with 10mM NHS-PEG4-Azide solution and incubated for 1h under gentle shaking to achieve NHS-PEG4-Azide labeling of cell surface proteins.

[0061] (4) Then add 100mM Tris-HCl buffer (pH 7.4) to quench the residual NHS-PEG4-Azide for 5 min to terminate the reaction.

[0062] (5) Next, add 1x TNTE lysis buffer (containing 50mM Tris-Cl pH 7.4, 1mM EGTA, 150mM NaCl, and 1% Triton X-100) and 1x protease inhibitor (Thermo Scientific, product #A32961), lyse cells on ice for 30 min, and then sonicate. (Plus sonication device) The cell lysate was centrifuged at 12000×g for 20 min at 4°C to obtain azide-labeled cell lysate and supernatant; the supernatant was transferred to a new tube and protein concentration was detected using a BCA kit (BioRad, products #500-0111 and #500-0112).

[0063] (6) Resuspend 100 μl of streptavidin magnetic beads (Thermo Scientific, product #65601) in 400 μl of PBS buffer, add 2 μl of 10 mM phosphine-PEG3-Biotin (EZ-Link Phosphine-PEG3-Biotin, Thermo Scientific, product #88901) to maintain a final concentration of 0.05 mM, and incubate at room temperature for 1 h to ensure that all streptavidin sites on the magnetic beads are completely coated with Phosphine-PEG3-Biotin. Wash 4 times with PBS buffer (pH 7.4) to obtain phosphine-biotin-streptavidin magnetic beads.

[0064] (7) Azide-labeled cell lysates were incubated with phosphine-biotin-streptavidin magnetic beads in PBS buffer at 37°C for 4 h to perform a bioorthogonal reaction between azide and phosphine, enriching azide-labeled cell surface proteins. After the reaction, magnetic beads containing cell surface proteins were obtained. The magnetic beads were washed three times with 150 mM NaCl solution or 1% SDS solution, and finally washed five times with PBS buffer (pH 7.4) to obtain magnetic beads containing cell surface proteins.

[0065] (8) Resuspend the magnetic beads containing cell surface proteins in 50 μL of elution buffer I (containing 2 M urea; 50 mM Tris-HCl pH 8.0, 1 mM DTT, and 10 μg / ml sequencing-grade trypsin (Thermo Fisher #90057)) and incubate at 30 °C for 60 min with stirring at 700 rpm. Transfer the supernatant to a new tube. Elute the remaining magnetic beads three times in the dark with 25 μL of elution buffer II (containing 50 mM Tris-HCl pH 8.0, 5 mM iodoacetamide, and 2 M urea), and then combine all eluents. Add an additional 250 ng of trypsin to the combined eluents and incubate overnight at 32 °C in the dark. Terminate the reaction by adding 6 μL of 10% formic acid solution (FA). The sample was desalted on a C18 pipette tip (Thermo Fisher, product #87784) and dried at VAC speed. The sample was then redissolved in 15 μL of 0.1% formic acid solution for LC-MS analysis.

[0066] To verify the characteristics of NHS-PEG4-Azide's labeling of cell surface proteins in step (4) of step 3 above, the following experiment was conducted:

[0067] Cells were labeled with 10 mM NHS-PEG4-Azide (Thermo Scientific, product #26130) diluted in PBS (pH 7.4) for 1 hour at 4°C. The reaction was terminated by washing three times with 100 mM Tris buffer (pH 7.4). Cells were then incubated with 10 μM DBCO-Cy 5.5 in 2 mL of culture medium for 10 min. Cells were then washed with ice-cold PBS, fixed with 2% formaldehyde and DAPI, and observed using a Nikon A1HD25 confocal microscope. Experimental results are shown below. Figure 2A As shown.

[0068] Depend on Figure 2A As can be seen, microscopic imaging of the DBCO-Cy5 signal shows that NHS-PEG4-Azide effectively and selectively modifies the cell surface, indicating that NHS-PEG4-Azide mainly labels surface-exposed proteins, consistent with the membrane impermeability of the PEG4 moiety.

[0069] Comparative Example 1:

[0070] This comparative example uses conventional sulfonyl-NHS-SS-Biotin to label and isolate cell surface proteins in A549 cells. The specific procedure is as follows: Figure 1 As shown, the experimental procedure is as follows:

[0071] 5×10 6 A549 cells were washed three times with ice-cold PBS (pH 7.4) and then incubated with 10 mM sulfonyl-NHS-SS-Biotin (Sulfo-NHS-SS-Biotin, Thermo Scientific, product #21331) / PBS (pH 7.4) at 4°C with gentle shaking for 1 hour. After labeling, the cells were washed three times with ice-cold 100 mM Tris buffer (pH 7.4) to terminate labeling and remove residual biotinylation reagent. Cell lysis was performed as described in Example 1 above, and the lysate was incubated with 100 μL streptavidin beads (Thermo Scientific, product #65601) at 4°C for 3 hours to separate biotinylated proteins. After separation, magnetic beads containing cell surface proteins were obtained. The magnetic beads were washed three times with 150 mM NaCl / 1% SDS solution and then five times with PBS (pH 7.4).

[0072] To examine the binding of endogenous biotin-associated proteins, lysates of control cells not labeled with NHS reagents were used. Western blotting using streptavidin-HRP showed that, under control conditions without NHS-PEG4-Azide and Sulfo-NHS-SS-Biotin labeling, the background of the BCAP method in Example 1 of this invention was significantly lower than that of the Sulfo-NHS-SS-Biotin method in Comparative Example 1 (e.g., ...). Figure 2B (As shown). Experiments suggest that the binding of endogenous biotin-associated proteins is minimized because the streptavidin site is occupied by phospho-polyethylene glycol-biotin before incubation with cell lysates. Notably, using the same amount of streptavidin magnetic beads, the BCAP method also enriched significantly more proteins (e.g., ...) than the Sulfo-NHS-SS-Biotin method. Figure 2B As shown in the figure, this indicates that the BCAP method of the present invention has higher efficiency and lower endogenous interference.

[0073] To confirm that the proteins were surface-exposed, A549 cells labeled with NHS-PEG4-Azide were treated with 0.25% trypsin to remove surface proteins. Cells were then lysed and subjected to a bioorthogonal reaction with phosphine-polyethylene glycol-biotin anchored to streptavidin beads. The experiment showed a significant reduction in proteins leading to BCAP separation (e.g., ...). Figure 2C (As shown).

[0074] The experiments in summary demonstrate that the BCAP method of this invention can efficiently and specifically separate surface-exposed proteins.

[0075] Example 2: LC-MS Analysis

[0076] In this embodiment, mass spectrometry was used to quantitatively compare the BCAP method of Example 1 with the direct Sulfo-NHS-SS-Biotin method.

[0077] As mentioned earlier, approximately 5×10 6 A549 cells were labeled with either NHS-PEG4-Azide or Sulfo-NHS-SS-Biotin reagent. After cell lysis, NHS-PEG4-Azide-labeled proteins were pulled down using phospho-biotin-streptavidin magnetic beads. In another case, proteins labeled with Sulfo-NHS-SS-Biotin were directly purified using streptavidin beads. Both methods were performed in two biological replicates. After a thorough washing step, the proteins were digested on the beads with trypsin. The obtained peptides were desalted and analyzed by LC-MS / MS using a label-free quantification method. The specific analytical procedure is as follows:

[0078] 1. Sample pretreatment:

[0079] The magnetic beads containing cell surface proteins were resuspended in 50 μL of elution buffer I (containing 2 M urea; 50 mM Tris-HCl pH 8.0, 1 mM DTT, and 10 μg / ml sequencing-grade trypsin (Thermo Fisher, product #90057)) and incubated at 30 °C for 60 min with stirring at 700 rpm. The supernatant was transferred to a new tube. The remaining magnetic beads were further eluted three times in the dark with 25 μL of elution buffer II (containing 50 mM Tris-HCl pH 8.0, 5 mM iodoacetamide, and 2 M urea), and all eluates were combined. An additional 250 ng of trypsin was added to the combined eluates, and the mixture was incubated overnight at 32 °C in the dark. The reaction was terminated by adding 6 μL of 10% formic acid solution (FA). The sample was desalted on a C18 pipette tip (Thermo Fisher, product #87784) and dried at VAC speed to obtain the sample, which was then redissolved in 15 μL of 0.1% formic acid solution.

[0080] 2. LC-MS / MS analysis:

[0081] Six μL of sample was loaded onto a Thermo Easy-Spray analytical column (75 μm inner diameter × 500 mm) C18 column equipped with an Easy-nLC 1200 chromatogram for coupling with a Thermo Q-Exative mass spectrometer. Each run was performed with a 125-minute gradient (5%–40% acetonitrile). The mass spectrometer was set to MS2 TopN mode with full MS / data correlation: mass analyzer coverage of 400–1600 m / z, mass resolution of 70,000 (m / z = 200), 35 NEC (normalized collision energy), 2.0 m / z separation window, and 15 s dynamic size exclusion.

[0082] The obtained raw spectral data were analyzed using Proteome Discoverer 2.2 software. The parameters were set as follows: maximum deletion fragmentation = 2, fixed modification = carbamoyl methyl / (C), variable modification = oxidation (0) and N-terminal acetylation (protein N-terminus), precursor mass tolerance = 10 ppm, and fragment mass tolerance = 0.02 Da.

[0083] 3. Staining of aging-related β-galactosidase:

[0084] The day before staining, 5×10 4Mouse fibroblasts (MEFs) were seeded into 6-well culture dishes. β-galactosidase staining was performed using the senescence β-galactosidase staining kit (Cell Signaling, product #9860) according to the manufacturer's instructions. Briefly, cells were washed with 1X PBS before adding 1 mL of 1X fixative. After incubating at room temperature for 10 minutes, cells were washed twice with 1X PBS. 1 mL of β-galactosidase staining solution was added to the wells, and the cells were incubated overnight at 37°C. The next day, the β-galactosidase activity indicated by blue in the cells was examined using an optical microscope.

[0085] 4. Immunofluorescence labeling and microscopic observation:

[0086] 12 hours before the experiment, 5×10 4 Mouse fibroblasts (MEFs) were seeded onto coverslips in 12-well culture dishes and cultured overnight at 37°C and 5% CO2. Cells were fixed with 2% paraformaldehyde / PBS at 4°C for 10 minutes. Next, cells were incubated directly with 1% BSA / PBS for 1 hour, or permeabilized with 0.1% Triton X-100 / PBS at 4°C for 10 minutes, followed by blocking with 1% BSA / PBS. Subsequently, cells were incubated overnight at 4°C with EHD2 primary antibody (Santa Cruz, product #sc-515458 1:100). After washing three times in 1% BSA / PBS, cells were incubated with secondary antibody (CST, product #8890S) in 1% BSA / PBS (1:400) at room temperature in the dark for 1.5 hours. After further washing, cells were fixed with DAPI (Thermo Fisher, product #62247) and observed using a Nikon A1HD25 confocal microscope.

[0087] The night before the experiment, 5×10 4 A549 cells were seeded on 35 mm confocal culture dishes (NESTBiotechnology, product #801001) and cultured at 37°C and 5% CO2. The next day, after removing the culture medium and washing in PBS, the cells were labeled for 1 hour at 4°C with 10 mM NHS-PEG4-Azide (ThermoScientific, product #26130) diluted in PBS (pH 7.4). The reaction was terminated by washing three times with 100 mM Tris buffer (pH 7.4). The cells were then incubated with 10 μM DBCO-Cy5.5 in 2 mL of culture medium for 10 minutes. The cells were then washed with ice-cold PBS, fixed with 2% formaldehyde and DAPI, and observed using a Nikon A1HD25 confocal microscope.

[0088] 5. Results

[0089] (1) Comparison of BCAP and direct Sulfo-NHS-SS-Biotin biotinylation method using label-free quantitative proteomics In this example, the number of proteins identified using the BCAP method of Example 1 and the direct Sulfo-NHS-SS-Biotin method and their enrichment of cellular components were analyzed.

[0090] Two BCAP replicates identified 509 and 506 proteins, respectively, with 501 proteins shared between the experiments. In contrast, the Sulfo-NHS-SS-Biotin experiments (which identified 249 and 217 proteins, respectively) shared 196 proteins (e.g., ...). Figure 3A As shown). The BCAP group recovered a relatively large amount of protein, which is consistent with the results of Western blotting (e.g., ...). Figure 2B (As shown). GO terminology annotations of the identified proteins revealed enrichment of cellular components associated with extracellular, membrane, adhesion, and junctional terms, indicating successful isolation of surface proteins (such as...) by both methods. Figure 3B (As shown in Table 1). It is noteworthy that 194 out of the 196 proteins identified by the Sulfo-NHS-SS-Biotin method were included in the BCAP method (e.g., ...). Figure 3C (As shown). Annotation analysis indicates that the BCAP method recovers more proteins from various compartments on the cell surface, including the plasma membrane, extracellular regions, and cell-cell adhesion junctions (such as...). Figure 3D (As shown). In summary, these results demonstrate the superior efficacy of the BCAP method of the present invention in the separation and recovery of cell surface proteins.

[0091] Table 1: The most representative GO term in the cellular components of each method

[0092]

[0093] Quantitative analysis is powerful in studying the kinetics of surface proteomes in different biological environments. The quantitative reproducibility of proteins identified in replicates using the BCAP and Sulfo-NHS-SS-Biotin methods was then compared. Label-free quantification showed similar abundance distributions of recovered proteins between replicates (e.g., ...). Figure 3E (As shown). Furthermore, both the BCAP and Sulfo-NHS-SS-Biotin methods demonstrated excellent quantitative reproducibility in both biological replicates, R... 2 The values ​​are 0.87 and 0.83 respectively. Figure 3E These results demonstrate that the BCAP method of this invention has good reliability in quantitative analysis.

[0094] (2) Analysis of surface proteins of senescent cells using the BCAP protocol

[0095] Cellular senescence is a process that permanently arrests cell proliferation. Senescent and proliferating cells exhibit significant differences in protein expression patterns (including cell surface proteins), which can serve as biomarkers and therapeutic targets. We used BCAP combined with label-free quantitative proteomics to identify differentially expressed surface proteins in senescent and proliferating cells. We adapted an established cellular senescence model: extended culture of primary mouse embryonic fibroblasts (MEFs). Generally, cells with a population doubling level (PDL) of 18–20 showed good proliferative capacity and could be used as proliferating MEFs (P). In contrast, cells with a PDL greater than 27 showed a slow growth rate and were used as senescent MEFs (S) (e.g., ...). Figure 6A (As shown). MEF(S) cells also exhibited higher senescence-associated β-galactosidase (SA-β-gal) activity than MEF(P), a significant characteristic of cellular senescence (e.g., Figure 6B (As shown).

[0096] For 5×10 6 Using the BCAP method, 434 and 428 proteins (such as...) were recovered from MEF(P) and MEF(S) cells, respectively. Figure 4A Interestingly, 426 proteins were shared between the two cell types, indicating no significant differences in the surface proteome between MEF(P) and MEF(S) cells. Of these proteins, over 53.5% and 17.6% were labeled as cell surface proteins in extracellular regions and plasma membranes, respectively (e.g., ...). Figure 4B (As shown).

[0097] Then, differential analysis was performed on the quantitative data of 426 proteins shared between MEF(P) and MEF(S) cells. This revealed 22 significantly different proteins (Benjamini-Hochberg adjusted p-value < 0.05) (e.g., Figure 4C Notably, elastin (Eln), an extracellular matrix (ECM) protein, appeared as a top protein with enhanced expression in MEF(P) (log2 fold change >5 and Benjamini-Hochberg adjusted p-value <0.001). This is consistent with previous observations that Eln expression decreases during cellular senescence, supporting the reliability of integrating BCAP with quantitative analysis.

[0098] Among the surface proteins upregulated in senescent MEFs, Western blot analysis using the BCAP method of this invention confirmed the presence of EHD2 (e.g., ...) on the surface of senescent MEF(S) cells. Figure 5A(As shown). EHD2 is a dynein-associated ATPase known to regulate cell membrane pit dynamics. In contrast, EHD2 signaling was not detected in surface proteins isolated from MEF(P) cells (e.g., Figure 5A (As shown). Then, this invention uses immunostaining to further examine the differences in EHD2 cell localization (e.g., ...). Figure 5B and Figure 5C (As shown). Without membrane permeation via Triton-X100, experiments observed stronger surface staining of EHD2 in MEF(S) cells (e.g., ...). Figure 5C (As shown). On the other hand, MEF(P) cells did not show EHD2 markers (e.g., without membrane permeation) in the absence of membrane permeation. Figure 5C (As shown). Quantitative analysis revealed a significantly higher number of EHD2-positive MEF(S) cells than MEF(P) cells in the absence of Triton-X100 permeabilization. In contrast, Triton-X100 permeabilization indicated that EHD2 was predominantly distributed in the cytoplasm of MEF(P) cells, and there was no significant difference in EHD2-positive cells between MEF(S) and MEF(P). These results demonstrate distinct EHD2 localization in MEF(S) and MEF(P) cells and validate the reliability of BCAP in analyzing differential surface proteins between two cell states.

[0099] In summary, this invention develops a bioorthogonal linkage-assisted purification method (BCAP) utilizing the azide-phosphine reaction, which exhibits lower background binding and higher efficiency in purifying cell surface proteins compared to direct labeling methods using NHS-SS-biotin; it also demonstrates good reproducibility when combined with label-free proteomics quantification. Therefore, the bioorthogonal linkage-assisted purification method (BCAP) of this invention is suitable for comparative analysis of surface proteins in cells under different conditions. This method was used to identify and validate EHD2 as a protein present in increased amounts on the surface of senescent MEFs.

Claims

1. A method for isolating and purifying cell surface proteins, comprising the following steps: Cells to be isolated were cultured, and an azide solution was added to the cells for incubation. The reaction was then terminated by adding Tris-HCl buffer to label the cell surface proteins with azides. Next, TNTE buffer and protease inhibitor were added to lyse the cells, and after centrifugation, azide-labeled cell lysates were obtained. Azide-labeled cell lysates were incubated with phospho-biotin-streptavidin magnetic beads to enrich azide-labeled cell surface proteins, resulting in magnetic beads containing cell surface proteins. in, The phosphine-biotin-streptavidin magnetic beads are obtained by incubating phosphine-polyethylene glycol-biotin with streptavidin magnetic beads and then washing them.

2. The method according to claim 1, further comprising: Magnetic beads containing cell surface proteins are added to elution buffer and incubated to elute the cell surface proteins from the magnetic beads.

3. The method according to claim 1 or 2, further comprising: Proteins isolated and enriched from cells are enzymatically digested to obtain peptides of cell surface proteins.

4. The separation and purification method according to claim 1, wherein, The cells to be separated include human non-small cell lung cancer A549 cells and / or mouse embryonic fibroblasts.

5. The separation and purification method according to claim 1, wherein, The azide solution is a 10 mM azide solution prepared with PBS buffer at pH 8.

0. The azide includes succinimide acrylate-polyethylene glycol-azide; The temperature for incubating the cells with azide solution was 4°C, and the incubation time was 1 hour.

6. The separation and purification method according to claim 1, wherein, The Tris-HCl buffer solution has a concentration of 100 mM and a pH of 7.

4. The reaction was terminated by adding Tris-HCl buffer for 5 minutes.

7. The separation and purification method according to claim 1, wherein, The TNTE buffer solution contains 50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Triton-X100, and 1 mM EDTA. Cells were lysed using an ice bath for 30 minutes.

8. The separation and purification method according to claim 1, wherein, Phosphine-polyethylene glycol-biotin and streptavidin magnetic beads were incubated in PBS buffer at room temperature for 1 hour.

9. The separation and purification method according to claim 1, wherein, The azide-labeled cell lysates were incubated with phospho-biotin-streptavidin magnetic beads at 37°C for 4 hours.

10. The separation and purification method according to claim 1 or 9, wherein, The washing after incubation was performed using PBS buffer with a pH of 7.

4.

11. The separation and purification method according to claim 2, wherein, The process of adding magnetic beads containing cell surface proteins to elution buffer, incubating, and then eluting includes: Add elution buffer I to the magnetic beads containing cell surface proteins and incubate for 1 hour. Then add elution buffer II to wash the magnetic beads and collect the supernatant.

12. The separation and purification method according to claim 11, wherein, The elution buffer I comprises: 2M urea, 50mM Tris-HCl at pH 8.0, 1mM DTT, and 10μg / ml sequencing-grade trypsin. The eluent II comprises: 50 mM Tris-HCl at pH 8.0, 5 mM iodoacetamide, and 2 M urea.

13. The application of the method for isolating and purifying cell surface proteins according to any one of claims 1 to 12 in the quantitative analysis of cell surface proteome.

Images