CN116102612A - Separation and purification method and application of cell surface protein - Google Patents

Abstract

The invention provides a method for separating and purifying cell surface proteins and application thereof. The method comprises the following steps: culturing to obtain cells to be separated, adding azide solution into the cells for incubation, and then adding Tris-HCl buffer solution to terminate the reaction so as to realize the labeling of cell surface protein azide; then adding TNTE buffer solution and protease inhibitor to lyse cells, and centrifuging to obtain azide-labeled cell lysate; the azide-labeled cell lysate is incubated with phosphine-biotin-streptavidin magnetic beads, and azide-labeled cell surface proteins are enriched to obtain magnetic beads containing the cell surface proteins. The method can effectively separate the cell surface protein, and has low background, high efficiency and good repeatability; can be applied to quantitative analysis of cell surface proteome.

Description

Separation and purification method and application of cell surface protein

Technical Field

The invention belongs to the technical field of protein purification, and relates to a separation and purification method and application of cell surface proteins.

Background

Cell surface proteins play a critical role in cell-to-cell recognition and cell-to-microenvironment interactions. Cell surface biotinylation has been one of the most commonly used surface proteome analysis methods, and at present, a method of directly capturing biotinylated cell surface proteins using streptavidin magnetic beads has been widely used, however, such conventional biotinylation method is affected by cell endogenous biotin-related proteins. Surface biotinylation has been widely used for analysis of cell proteomes associated with plasma membranes. However, the workflow is disturbed by cytoplasmic biotin-related proteins that compete for streptavidin binding during purification, interfering with the accuracy and efficiency of the quantitative analysis.

Disclosure of Invention

Based on the defects of the prior art, an object of the invention is to provide a method for separating and purifying cell surface proteins. Another object of the present invention is to provide the use of the method for the isolation and purification of cell surface proteins for the quantitative analysis of the cell surface proteome.

The separation and purification method of the cell surface protein of the present invention is a bio-orthogonal ligation-assisted purification method (Bioorthogonal Conjugation-Assisted Purification, BCAP) in which the characteristic of chemically selective ligation of Staudinger (Staudinger) is used to label and separate cell surface related proteins, thereby minimizing the interference of endogenous biotin-related proteins. In the BCAP workflow, the cell surface exposed proteins were first labeled with NHS-PEG4-Azide, and then TNTE was added to lyse the cells after termination of the reaction with 100nM Tris-HCl to obtain lysates. Simultaneously, the Phosphine-PEG3-Biotin and the streptavidin magnetic beads are incubated together, so that all streptavidin sites on the magnetic beads are completely coated by the Phosphine-PEG 3-Biotin. The coated phosphine-biotin-streptavidin magnetic beads were incubated with cell lysates, and cell surface proteins labeled with NHS-PEG4-Azide were enriched by bio-orthogonal reaction between Azide and phosphine. The method of the invention can effectively separate the cell surface protein and has high repeatability.

Specifically, in one aspect, the present invention provides a method for separating and purifying a cell surface protein, comprising the steps of:

culturing to obtain cells to be separated, adding azide solution into the cells for incubation, and then adding Tris-HCl buffer solution to terminate the reaction so as to realize the labeling of cell surface protein azide;

then adding TNTE buffer solution and protease inhibitor to lyse cells, and centrifuging to obtain azide-labeled cell lysate;

incubating the azide-labeled cell lysate with phosphine-biotin-streptavidin magnetic beads, and enriching azide-labeled cell surface proteins to obtain magnetic beads containing the cell surface proteins;

wherein the phosphine-biotin-streptavidin magnetic beads are obtained by incubating and washing phosphine-triethylene glycol-biotin and streptavidin magnetic beads.

According to a specific embodiment of the present invention, the method for separating and purifying a cell surface protein of the present invention further comprises: and adding the magnetic beads containing the cell surface proteins into the eluent for incubation, eluting, and eluting the cell surface proteins from the magnetic beads.

According to a specific embodiment of the present invention, the method for separating and purifying a cell surface protein of the present invention further comprises: the peptide of the cell surface protein is obtained by enzymolysis of the protein separated and enriched from the cells.

In the above separation and purification method, preferably, the cells to be separated include human non-small cell lung cancer a549 cells and/or mouse embryo fibroblasts; but is not limited thereto.

In the above separation and purification method, preferably, the azide solution is an azide solution having a concentration of 10mM prepared by PBS buffer having a pH of 8.0.

In the above separation and purification method, preferably, the Azide includes succinimide-tetra polyethylene glycol-Azide (NHS-PEG 4-Azide); but is not limited thereto.

In the above separation and purification method, preferably, the Tris-HCl buffer has a concentration of 100mM and a pH of 7.4.

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

In the above separation and purification method, preferably, the azide solution is added to the cells to perform the incubation reaction at a temperature of 4℃for a period of 1 hour.

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

In the above separation and purification method, preferably, the cells are lysed by using an ice bath for 30min.

In the above separation and purification method, preferably, the phosphine-tripolyglycol-biotin solution is a phosphine-tripolyglycol-biotin solution having a concentration of 0.05mM prepared by using PBS buffer having a pH of 7.4. In the above separation and purification method, preferably, the temperature at which the phosphine-tripolyethylene glycol-biotin and streptavidin magnetic beads are incubated is room temperature, and the incubation time is 1h.

In the above separation and purification method, preferably, the temperature of incubation of azide-labeled cell lysate with phosphine-biotin-streptavidin magnetic beads is 37℃and the incubation time is 4 hours.

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

According to a specific embodiment of the present invention, in the method for separating and purifying a cell surface protein of the present invention, the process of adding magnetic beads containing a cell surface protein to an eluent, incubating and eluting comprises: the magnetic beads containing the cell surface proteins were incubated for 1 hour by adding eluent I, then washed by adding eluent II, and the supernatant was collected.

In the above separation and purification method, preferably, the eluent I includes: 2M Urea, 50mM Tris-HCl pH 8.0, 1mM DTT, 10. Mu.g/mL sequencing grade trypsin.

In the above separation and purification method, preferably, the eluent II includes: 2M urea, 50mM Tris-HCl pH 8.0, 5mM iodoacetamide (iodoacetamide).

On the other hand, the invention also provides a phosphine-biotin-streptavidin magnetic bead used for the separation and purification method of the cell surface protein, which is obtained by incubating and washing the phosphine-tri-polyethylene glycol-biotin and the streptavidin magnetic bead.

In another aspect, the invention also provides the use of an azide as a marker for labelling a cell surface protein. Preferably, the azide comprises an acrylic succinimide-tetra polyethylene glycol-azide.

On the other hand, the invention also provides application of the separation and purification method of the cell surface protein in quantitative analysis of a cell surface proteome.

The invention has the beneficial effects that: the biological orthogonal connection auxiliary purification method (BCAP) is adopted to separate and purify the cell surface protein, so that the cell surface protein can be effectively separated, and the method has low background, high efficiency and good repeatability; in addition, the method for separating and purifying the cell surface protein can be suitable for the comparative analysis of the surface protein in cells under different conditions, can realize efficient and repeatable cell surface proteome analysis, is applied to the difference of the surface membrane protein of mouse fibroblast (MEF) which is proliferated and aged, and also discovers that the cell membrane localization of EHD2 in aged MEF is enhanced.

Drawings

FIG. 1 is a comparison of the workflow of isolating cell surface proteins using the bioorthogonal ligation-assisted purification method (BCAP) of the invention and the conventional sulfo-NHS-SS-biotin method (the BCAP method of the invention is intended to minimize competitive binding of endogenous biotin-related proteins).

FIG. 2A is a 20 μm scale of detection of A549 cells with or without NHS-PEG4-Azide markers using DBCO-Cy5 and DAPI.

FIG. 2B is a Western blot analysis of cell surface preparation (wherein A549 cells were treated with the indicated labeling procedure prior to cell lysis; whole cell lysates were incubated with streptavidin or phospho-biotin-streptavidin beads; biotin blotting of isolated proteins with HRP-streptavidin).

FIG. 2C is a Western blot analysis of BCAP in the absence or presence of 0.25% trypsin/EDTA treatment (biotin blotting of isolated proteins using HRP-streptavidin).

FIG. 3A is a Venn diagram showing the number of surface-associated proteins identified by two biological replicates of a single process.

FIG. 3B is a Venn diagram showing the overlap of proteins identified by two methods after binding data from two replicates.

Fig. 3C is a graph of the first five enrichment classes of GO enrichment analysis of cellular compartments of proteins identified by two methods.

FIG. 3D is a graph of the amount of protein in the indicated categories of GO enrichment analysis of cellular compartments of proteins identified by two methods.

FIG. 3E is a comparative plot of quantitative reproducibility of proteins isolated by the Sulfo-NHS-SS-Biotin method (left panel) and the BCAP method (right panel) (histograms of log 10-converted abundances of two biologically repeated isolated proteins are distributed along the x and y axes, respectively).

Fig. 4A is a comparative graph of surface-associated proteins isolated from proliferating and senescent MEFs by BCAP method (which combines the results of two biological replicates).

Fig. 4B is a graph of GO enrichment analysis of cellular compartments of proteins identified from proliferating and senescent MEFs using BCAP methods.

FIG. 4C is a volcanic plot of surface proteome difference analysis between proliferating and senescent MEFs (vertical and horizontal dashed lines demarcate an absolute fold change of 2 and an adjusted p-value of 0.05, respectively).

FIG. 5A is a Western blot of anti-EHD 2 antibody detection of total cell lysates of proliferating and senescent MEFs and protein enriched by the BCAP method.

FIG. 5B is a graph of the percentage of cells of the immunomarker positive surface markers of EHD2 in the absence or presence of Triton X-100 mediated permeabilization in proliferating (P) and senescent (S) MEFs (results describe mean.+ -. SEM from 3 replicates, each quantifying >50 cells).

FIG. 5C is a representative graph of immunolabeling of EHD2 in proliferation (P) and senescence (S) MEFs in the absence or presence of Triton X-100 mediated permeabilization.

FIG. 6A is a graph of culture propagation time and corresponding Population Doubling Level (PDL) for MEFs.

Fig. 6B is a representative plot of senescence-associated beta-galactosidase (SA-beta-gal) staining of proliferative and senescent MEFs (the percentage of positive cells is quantified in the right plot (the results describe mean ± SEM, n=3)).

Detailed Description

The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention.

The starting reagent materials in each example are commercially available, unless otherwise noted. The processes not specified in the examples are carried out according to the usual operating conditions in the art or as recommended by the instructions of the manufacturer of the apparatus.

Example 1:

1. cell culture:

human non-small cell lung carcinoma (NSCLC) A549 (ATCC CCL-185) cells were cultured in DMEM medium containing 10% FBS, 2mM L-glutamine and 100. Mu.g/ml penicillin-streptomycin (Thermo Fisher).

2. Isolation and culture of Mouse Embryonic Fibroblasts (MEFs):

the study used C57BL/6J mice cultured in cycles at 12 hours each in the dark and in all mice experiments were conducted according to the protocols approved by the animal research ethics group committee of the university of hong Kong City and the government health agency of hong Kong.

To obtain E13.5 mouse embryos, male and female mice 8 to 15 weeks old were mated. Female mice pregnant with E13.5 were anesthetized with 4% isoflurane by inhalation, and embryos were then removed from the ovaries. After taking the embryo, immediately breaking the head to kill the pregnant mouse; the embryo tissue was then minced with a knife in ice PBS (pH 7.4) buffer. Thereafter, the tissue fragments were incubated in 0.25% trypsin for 15 minutes at 37℃and then centrifuged at 1500rpm for 5 minutes at 4 ℃. 2mL 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 dried at 75cm ² Culturing in culture flask. To determine the cumulative population doublings, the initial and final viable cell counts were recorded as well as the time to 90% confluency. PDL is calculated by the following formula:

PDL＝3.32(logXf-logXi)+S

in the formula, S represents PDL at the start of culture, xf represents the final viable cell count, and Xi is the initial inoculated cell count.

3. Cell surface protein labeling and isolation (main flow scheme is shown in fig. 1):

(1) Will be 5X 10 ⁶ Individual a549 cells or Mouse Embryonic Fibroblasts (MEFs) were washed 3 times with ice PSB (ph 7.4) buffer.

(2) Succinimide-tetra polyethylene glycol-Azide (NHS-PEG 4-Azide, thermo Scientific, product # 26130) was added to PBS (pH 7.4) buffer to make a 10mM NHS-PEG4-Azide solution.

(3) A549 cells or Mouse Embryonic Fibroblasts (MEFs) were mixed with 10mM NHS-PEG4-Azide solution at 4deg.C and incubated for 1h with gentle shaking to effect labeling of the cell surface proteins by NHS-PEG 4-Azide.

(4) The reaction was then quenched by the addition of 100mM Tris-HCl buffer (pH 7.4) for 5min with residual NHS-PEG 4-Azide.

(5) Then 1 XTNTE lysate (containing 50mM Tris-Cl pH7.4, 1mM EGTA,150mM NaCl, 1% Triton X-100) and 1 Xprotease inhibitor (Thermo Scientific, product #A 32961) were added thereto and the cells were lysed in an ice bath for 30min, followed by sonication [ ]

plus sonication device), the cell lysate was centrifuged at 12000×g for 20min at 4 ℃ to give azide-labeled cell lysate and supernatant; the supernatant was transferred to a new tube and protein concentration was measured using BCA kit (BioRad, products #500-0111 and # 500-0112).

(6) 100 μl of streptavidin beads (Thermo Scientific, product # 65601) were resuspended in 400ul of PBS buffer, 2 μl of 10mM Phosphine-tripolyethylene glycol-Biotin (EZ-Link Phosphine-PEG3-Biotin, thermo Scientific, product # 88901) was added and the final concentration was kept at 0.05mM, and incubated at room temperature for 1h to allow all streptavidin sites on the beads to be completely coated with Phosphine-PEG3-Biotin, and washed 4 times with PBS buffer (pH 7.4) to give Phosphine-Biotin-streptavidin beads.

(7) Incubating the azide-labeled cell lysate with phosphine-biotin-streptavidin magnetic beads in PBS buffer at 37 ℃ for 4h to perform a bio-orthogonal reaction between azide and phosphine, enriching for azide-labeled cell surface proteins; after the reaction, magnetic beads containing cell surface proteins were obtained, and the magnetic beads were washed 3 times with 150mM NaCl solution or 1% SDS solution, and finally washed 5 times with PBS buffer (pH 7.4) to obtain magnetic beads containing cell surface proteins.

(8) The beads containing the cell surface proteins were resuspended in 50. Mu.L of elution buffer I (2M urea; 50mM Tris-HCl pH 8.0, 1mM DTT, 10. Mu.g/ml sequencing grade trypsin (Thermo Fisher # 90057)) and incubated at 30℃for 60min with stirring at 700rpm and the supernatant transferred to a new tube. The remaining beads were further eluted 3 times in the dark with 25. Mu.L of elution buffer II (50 mM Tris-HCl pH 8.0, 5mM iodoacetamide, 2M urea) and all the eluates were pooled. An additional 250ng of trypsin was added to the combined eluates, which were then incubated overnight at 32℃and protected from light. The reaction was stopped by adding 6. Mu.l of 10% formic acid solution (FA). After desalting on a C18 tip (Thermo Fisher, product # 87784) and drying at VAC speed, a sample was obtained, which was redissolved in 15. Mu.L of 0.1% formic acid solution for LC-MS analysis.

To verify the characteristic of the NHS-PEG4-Azide labeling of cell surface proteins in step (4) above in step 3, the following experiment was performed:

cells were labeled with 10mM NHS-PEG4-Azide (Thermo Scientific, product # 26130) diluted in PBS (pH 7.4) for 1 hour at 4 ℃. The reaction was stopped by washing 3 times with 100mM Tris buffer (pH 7.4). The cells were then incubated with 10. Mu.M DBCO-Cy5.5 in 2mL of medium for 10 min. Cells were then washed with ice-cold PBS, fixed with 2% formaldehyde and DAPI, and then observed with a nikon A1HD25 confocal microscope. The experimental results are shown in FIG. 2A.

As can be seen from fig. 2A: microscopic imaging of the DBCO-Cy5 signal showed that NHS-PEG4-Azide was effectively and selectively modified to the cell surface, thus indicating that NHS-PEG4-Azide primarily labels the surface-exposed proteins, consistent with the membrane impermeability of the PEG4 moiety.

Comparative example 1:

the comparative example adopts conventional Sulfo-NHS-SS-Biotin (Sulfo-NHS-SS-Biotin) to mark and separate the cell surface protein of A549 cells, and the specific flow is shown in figure 1, and the experimental process is as follows:

will be 5X 10 ⁶ Individual a549 cells were washed 3 times with ice-cold PBS (pH 7.4) and then incubated with 10mM Sulfo-NHS-SS-Biotin (sulfoo-NHS-SS-Biotin, thermo Scientific, product # 21331)/PBS (pH 7.4) at 4 ℃ for 1 hour with gentle shaking. After labelling, the cells were washed 3 times with ice-cold 100mM Tris buffer (pH 7.4) to terminate labelling and remove residual biotinylated reagent. Cell lysis was performed as described in example 1 above, and lysates were incubated with 100. Mu.L of streptavidin beads (Thermo Scientific, product # 65601) at 4℃for 3 hours to isolate biotinylated proteins, after which the beads containing cell surface proteins were obtained, and the beads were washed 3 times with 150mM NaCl/1% SDS solution and 5 times with PBS (pH 7.4).

To examine the binding of endogenous biotin-related proteins, lysates of control cells not labeled with NHS reagent were used. Western blotting using streptavidin-HRP showed that the background of the BCAP method of example 1 of the present invention was significantly lower than that of the Sulfo-NHS-SS-Biotin method of comparative example 1 in the control condition without NHS-PEG4-Azide and Sulfo-NHS-SS-Biotin labeling (as shown in FIG. 2B). Experiments have concluded that streptavidin sites minimize the binding of endogenous biotin-related proteins, since they are already occupied by phosphine-tripolyethylene glycol-biotin prior to incubation with cell lysates. Notably, the BCAP method also enriches significantly more protein than Sulfo-NHS-SS-Biotin using the same amount of streptavidin beads (as shown in fig. 2B), indicating that the BCAP method of the invention is more efficient and has less endogenous interference.

To confirm that the protein was surface exposed, the NHS-PEG4-Azide labeled a549 cells were treated with 0.25% trypsin to remove surface protein, then the cells were lysed and bio-orthogonal reacted with phosphine-tri-polyethylene glycol-biotin anchored to streptavidin magnetic beads, which experiments showed a significant reduction in BCAP separation (as shown in fig. 2C).

The experiment in summary shows that: the BCAP method of the present invention is capable of efficiently and specifically isolating surface-exposed proteins.

Example 2: LC-MS analysis

In this example, the BCAP method of example 1 was quantitatively compared with the direct Sulfo-NHS-SS-Biotin method using mass spectrometry.

As previously mentioned, about 5X 10 is employed ⁶ The individual A549 cells were labeled with NHS-PEG4-Azide or Sulfo-NHS-SS-Biotin reagent. After cell lysis, NHS-PEG4-Azide labeled proteins were pulled down using magnetic beads with phosphine-biotin-streptavidin. In another case, the protein labeled by Sulfo-NHS-SS-Biotin was directly purified by streptavidin beads. Both methods were performed in duplicate biological replicates. After a number of washing steps, proteins were digested with trypsin on the beads. The peptides obtained were desalted by LC-MS/MS using label-free quantification method. The specific analysis process is as follows:

1. sample pretreatment:

the beads containing the cell surface proteins were resuspended in 50. Mu.L of elution buffer I (2M urea; 50mM Tris-HCl pH 8.0, 1mM DTT, 10. Mu.g/ml sequencing grade trypsin (Thermo Fisher, product # 90057)) and incubated at 30℃for 60min with stirring at 700rpm and the supernatant transferred to a new tube. The remaining beads were further eluted 3 times in the dark with 25. Mu.L of elution buffer II (50 mM Tris-HCl pH 8.0, 5mM iodoacetamide, 2M urea) and all the eluates were pooled. An additional 250ng of trypsin was added to the combined eluates, which were then incubated overnight at 32℃and protected from light. The reaction was stopped by adding 6. Mu.l of 10% formic acid solution (FA). After desalting on a C18 tip (Thermo Fisher, product # 87784) and drying at VAC speed, a sample was obtained, which was redissolved in 15. Mu.L of 0.1% formic acid solution.

2. LC-MS/MS analysis:

mu.L of the sample was loaded onto a Thermo Easy-Spray analysis column (75 μm inner diameter. Times.500 mm) C18 chromatography column with Easy-nLC 1200 chromatography in combination with a Thermo Q-Exactive mass spectrometer. Each run was run with a gradient of 125 minutes (5% to 40% acetonitrile). The mass spectrometer was set to MS2 TopN mode for complete MS/data correlation: mass analyzer in the m/z range of 400-1600, mass resolution 70000 (m/z=200), 35NEC (normalized collision energy), 2.0m/z separation window, 15s dynamic exclusion.

The raw data obtained were analyzed by software Proteome discover 2.2. The parameters were set as follows: maximum deletion cleavage = 2, fixed modification = carbamoylmethyl/(C), variable modification = oxidation (0) and N-terminal acetylation (protein N-terminal), precursor mass tolerance = 10ppm, and fragment mass tolerance = 0.02Da.

3. Aging-related beta-galactosidase staining:

the day before staining, 5X 10 ⁴ Individual mouse fibroblasts (MEFs) were inoculated into 6-well dishes. Beta-galactosidase staining was performed using an aging beta-galactosidase staining kit (Cell Signaling, product # 9860) according to the manufacturer's instructions. Briefly, cells were rinsed with 1 XPBS prior to the addition of 1mL of 1 Xfixative. After incubation for 10 min at room temperature, the cells were rinsed twice with 1X PBS. 1mL of beta-galactosidase staining solution was added to the wells and then incubated overnight at 37 ℃. The following day, cells were examined for blue-indicated β -galactosidase activity using an optical microscope.

4. Immunofluorescence labeling and microscopy:

12 hours before the experiment, 5X 10 ⁴ Individual mouse fibroblasts (MEFs) were inoculated onto coverslips of 12-well petri dishes and incubated at 37 ℃ with 5% co ₂ Under culture ofAnd (5) at night. Cells were fixed with 2% paraformaldehyde/PBS for 10 min at 4 ℃. Next, the cells were either incubated directly with 1% BSA/PBS for 1 hour or permeabilized with 0.1% Triton X-100/PBS for 10 minutes at 4℃and then blocked with 1% BSA/PBS. Subsequently, EHD2 primary antibodies (Santa Cruz, product #sc-515458 1:100) were incubated with the cells overnight at 4 ℃. After 3 washes in 1% BSA/PBS, the cells were incubated with secondary antibody (CST, product # 8890S) in 1% BSA/PBS (1:400) for 1.5 hours at room temperature in the dark. After further washing, the cells were fixed with DAPI (Thermo Fisher, product # 62247) and observed with a Nikon A1HD25 confocal microscope.

Evening prior to the experiment, 5X 10 ⁴ Individual a549 cells were seeded on 35mm confocal petri dishes (NEST Biotechnology, product # 802001) and incubated at 37 ℃ with 5% co ₂ Is cultured. The following day, after removal of the medium and washing in PBS, the cells were labeled with 10mM NHS-PEG4-Azide (Thermo Scientific, product # 26130) diluted in PBS (pH 7.4) for 1 hour at 4 ℃. The reaction was stopped by washing 3 times with 100mM Tris buffer (pH 7.4). The cells were then incubated with 10. Mu.M DBCO-Cy5.5 in 2mL of medium for 10 min. Cells were then washed with ice-cold PBS, fixed with 2% formaldehyde and DAPI, and then observed with a nikon A1HD25 confocal microscope.

5. Results

(1) Quantitative proteomics comparison of BCAP with direct sulfoo-NHS-SS-Biotin biotinylation method without labelling in this example, the number of proteins identified using the BCAP method of example 1 and the direct sulfoo-NHS-SS-Biotin method and their enrichment of cellular components were analyzed.

Two BCAP replicates identified 509 and 506 proteins, respectively, with 501 proteins shared between experiments. In contrast, the Sulfo-NHS-SS-Biotin experiment (which identified 249 and 217 proteins, respectively) shared 196 proteins (as shown in FIG. 3A). The number of recovered proteins in BCAP group was high, which is consistent with western blotting results (as shown in fig. 2B). The GO term annotation of the identified proteins revealed an enrichment of cellular components related to extracellular, membrane, adhesion and ligation terms, indicating successful isolation of surface proteins by both methods (as shown in FIG. 3B, table 1). Notably, 194 of 196 proteins identified by the Sulfo-NHS-SS-Biotin method were incorporated into the BCAP method (as shown in FIG. 3C). Annotation analysis showed that BCAP methods recovered more protein from various compartments of the cell surface, including plasma membrane, extracellular region, and cell-cell adhesion linkage (as shown in fig. 3D). Taken together, these results demonstrate the superior efficacy of the BCAP method of the invention in isolating and recovering cell surface proteins.

TABLE 1 most representative GOterm in the cellular fraction of each method

Quantitative analysis is powerful in studying the kinetics of surface proteomics in different biological environments. Then, quantitative reproducibility of the proteins identified in the repetition of the BCAP and Sulfo-NHS-SS-Biotin methods was compared. Label-free quantification showed similar abundance distribution of recovered protein between replicates (as shown in fig. 3E). In addition, BCAP and Sulfo-NHS-SS-Biotin methods have excellent quantitative reproducibility in both biological replicates, R ² The values were 0.87 and 0.83, respectively (fig. 3E). These results indicate that the BCAP method of the present invention has good reliability in quantitative analysis.

(2) Analysis of surface proteins of senescent cells using BCAP protocol

Cell senescence is a process that imparts a permanent retardation to cell proliferation. Senescent cells and proliferating cells exhibit significant differences in protein expression patterns (including cell surface proteins), and can be used as biomarkers and therapeutic targets. Differential surface proteins of senescent and proliferating cells were identified using BCAP binding label-free quantitative proteomics. The established cell senescence model was adapted: expanded culture of primary Mouse Embryonic Fibroblasts (MEFs). In general, cells with Population Doubling Levels (PDL) of 18 to 20 show good proliferation capacity and can be used as proliferating MEFs (P). In contrast, cells with PDL exceeding 27 showed slow growth rate and were used as senescent MEF (S) (as shown in fig. 6A). MEF (S) cells also showed higher senescence-associated β -galactosidase (SA- β -gal) activity than MEF (P), a significant feature of cell senescence (as shown in fig. 6B).

For 5X 10 ⁶ The BCAP method was applied to individual cells, and 434 and 428 proteins were recovered from MEF (P) and MEF (S) cells, respectively (fig. 4A). Interestingly, 426 protein was shared between the two cell types, indicating that there was no significant difference between the surface proteomes of MEF (P) and MEF (S) cells. Of these proteins, more than 53.5% and 17.6% were annotated as cell surface proteins in the extracellular region and plasma membrane, respectively (as shown in fig. 4B).

Differential analysis was then performed on quantitative data of 426 proteins shared between MEF (P) and MEF (S) cells. This revealed 22 significantly different (Benjamini-Hochberg adjusted p-value < 0.05) proteins (see fig. 4C). Notably, elastin (Eln) is an extracellular matrix (ECM) protein that appears as a top protein (top protein) of enhanced expression in MEF (P) (log 2 fold change >5 and Benjamini-Hochberg adjusts P-value < 0.001). This is consistent with previous observations that Eln expression was reduced during cell senescence, supporting reliability of integration of BCAP with quantitative analysis.

Western blot after purification of BCAP method of the present invention demonstrates EHD2 presence on the cell surface of senescent MEF (S) among surface proteins upregulated in senescent MEF (as shown in fig. 5A). EHD2 is an dynein-related atpase known to have a function of regulating cell membrane pit dynamics. In contrast, no EHD2 signal was detected in the surface proteins isolated from MEF (P) cells (as shown in fig. 5A). The present invention then applies immunostaining to further examine the differences in EHD2 cell localization (as shown in fig. 5B and 5C). In the absence of membrane permeabilization by Triton-X100, the surface staining of EHD2 in MEF (S) cells was experimentally observed to be stronger (as shown in FIG. 5C). On the other hand, MEF (P) cells did not show EHD2 labeling without membrane permeabilization (as shown in fig. 5C). Quantitative analysis showed that EHD2 positive MEF (S) cells were significantly more than MEF (P) cells without Triton-X100 permeabilization. In contrast, triton-X100 permeabilization indicated that EHD2 was predominantly distributed in the cytoplasm in MEF (P) cells, and that there was no significant difference in EHD2 positive cells between MEF (S) and MEF (P). These results demonstrate different EHD2 localization in MEF (S) and MEF (P) cells and verify the reliability of BCAP in analyzing the differential surface proteins between the two cell states.

In summary, the present invention developed a bio-orthogonal ligation-assisted purification method (BCAP) using azide-phosphine reaction with lower background binding and higher efficiency in purifying cell surface proteins compared to a direct labeling method using NHS-SS-biotin; it has good reproducibility in combination with label-free proteomic quantification. Thus, the bioorthogonal ligation-assisted purification method (BCAP) of the invention is suitable for comparative analysis of surface proteins in cells under different conditions. Using this method EHD2 was identified and validated as a protein with increased presence on the surface of senescent MEFs.

Claims (15)

1. A method for separating and purifying a cell surface protein, comprising the steps of:

culturing to obtain cells to be separated, adding azide solution into the cells for incubation, and then adding Tris-HCl buffer solution to terminate the reaction so as to realize the labeling of cell surface protein azide;

then adding TNTE buffer solution and protease inhibitor to lyse cells, and centrifuging to obtain azide-labeled cell lysate;

incubating the azide-labeled cell lysate with phosphine-biotin-streptavidin magnetic beads, and enriching azide-labeled cell surface proteins to obtain magnetic beads containing the cell surface proteins;

wherein the phosphine-biotin-streptavidin magnetic beads are obtained by incubating and washing phosphine-triethylene glycol-biotin and streptavidin magnetic beads.

2. The method of claim 1, the method further comprising:

and adding the magnetic beads containing the cell surface proteins into the eluent for incubation, eluting, and eluting the cell surface proteins from the magnetic beads.

3. The method of claim 1 or 2, further comprising:

the peptide of the cell surface protein is obtained by enzymolysis of the protein separated and enriched from the cells.

4. The method of claim 1, wherein the cells to be isolated comprise human non-small cell lung cancer a549 cells and/or mouse embryonic fibroblasts.

5. The method of claim 1, wherein the azide solution is an azide solution at a concentration of 10mM prepared with PBS buffer at pH 8.0;

preferably, the azide comprises an acrylic succinimide-tetra polyethylene glycol-azide;

preferably, the temperature at which the incubation reaction is performed by adding azide solution to the cells is 4℃and the incubation time is 1h.

6. The method according to claim 1, wherein the Tris-HCl buffer has a concentration of 100mM and a pH of 7.4;

preferably, the reaction is stopped by adding Tris-HCl buffer for 5min.

7. The method according to claim 1, wherein the TNTE buffer comprises 50mM Tris-HCl pH7.4, 150mM NaCl, 1% Triton-X100 and 1mM EDTA;

preferably, the cells are lysed using an ice bath for 30min.

8. The method for separating and purifying according to claim 1, wherein the temperature at which the phosphine-tripolyethylene glycol-biotin and streptavidin magnetic beads are incubated in PBS buffer is room temperature, and the incubation time is 1h.

9. The method of claim 1, wherein the azide-labeled cell lysate is incubated with the phosphine-biotin-streptavidin magnetic beads at 37 ℃ for 4 hours.

10. The method according to claim 1 or 9, wherein the incubation is followed by washing with a PBS buffer having a pH of 7.4.

11. The separation and purification method according to claim 2, wherein the process of incubating and eluting the magnetic beads containing the cell surface proteins with the eluent comprises:

the magnetic beads containing the cell surface proteins were incubated for 1 hour by adding eluent I, then washed by adding eluent II, and the supernatant was collected.

12. The method for separating and purifying according to claim 11, wherein the eluent I comprises: 2M urea, 50mM Tris-HCl pH 8.0, 1mM DTT, 10. Mu.g/ml sequencing grade trypsin;

the eluent II comprises the following components: 50mM Tris-HCl pH 8.0, 5mM iodoacetamide, 2M urea.

13. A phosphine-biotin-streptavidin magnetic bead for use in the method of any one of claims 1-12, which is obtained after incubating and washing the phosphine-tripolyglycol-biotin with the streptavidin magnetic bead.

14. The use of an azide as a label for labelling a cell surface protein; preferably, the azide comprises an acrylic succinimide-tetra polyethylene glycol-azide.

15. Use of the method for the isolation and purification of a cell surface protein according to any one of claims 1 to 12 for the quantitative analysis of a cell surface proteome.

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