Search

CN-116102612-B - Separation and purification method and application of cell surface protein

CN116102612BCN 116102612 BCN116102612 BCN 116102612BCN-116102612-B

Abstract

The invention provides a method for separating and purifying cell surface proteins and application thereof. The method comprises the steps of culturing cells to be separated, adding azide solution into the cells for incubation reaction, adding Tris-HCl buffer solution for stopping reaction, achieving labeling of cell surface protein azide, adding TNTE buffer solution and protease inhibitor for cracking the cells, 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 the magnetic beads containing the cell surface proteins. The method can effectively separate the cell surface protein, has low background, high efficiency and good repeatability, and can be applied to quantitative analysis of the cell surface proteome.

Inventors

  • ZHANG LIANG
  • LIU GUOPAN
  • MA HAIYING

Assignees

  • 香港城市大学深圳研究院

Dates

Publication Date
20260508
Application Date
20211109

Claims (13)

  1. 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. 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. 3. The method of claim 1 or 2, further comprising: and (3) carrying out enzymolysis on the protein obtained by separating and enriching from the cells to obtain peptide fragments of the cell surface protein.
  4. 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. 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; Wherein the azide comprises an acrylic acid succinimide-tetrapolyethylene glycol-azide; Wherein the temperature of the incubation reaction by adding azide solution to the cells was 4 ℃ and the incubation time was 1h.
  6. 6. The method according to claim 1, wherein the Tris-HCl buffer has a concentration of 100mM and a pH of 7.4; wherein, the time for stopping the reaction by adding Tris-HCl buffer solution is 5min.
  7. 7. The method according to claim 1, wherein the TNTE buffer contains 50mM Tris-HCl pH 7.4, 150mM NaCl, 1% Triton-X100 and 1mM EDTA; Wherein, the cells are lysed by ice bath for 30min.
  8. 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. 9. The method of claim 1, wherein the azide-labeled cell lysate is incubated with the phosphine-biotin-streptavidin magnetic beads at a temperature of 37 ℃ for a period of 4 hours.
  10. 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. 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. 12. The method according to claim 11, wherein the eluent I comprises 2M urea, 50mM Tris-HCl with pH of 8.0, 1mM DTT and 10 μg/ml sequencing grade trypsin; The eluent II comprises 50mM Tris-HCl with pH of 8.0, 5mM iodoacetamide and 2M urea.
  13. 13. The use of the method for the separation and purification of a cell surface protein according to any one of claims 1 to 12 for quantitative analysis of a 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, cell surface exposed proteins were first labeled with NHS-PEG4-Azide, and then cells were lysed to obtain lysates by addition of TNTE after termination of the reaction with 100nM Tris-HCl. Meanwhile, phosphine-PEG3-Biotin and streptavidin magnetic beads are incubated together, so that all streptavidin sites on the magnetic beads are completely coated by 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 invention, the method for separating and purifying the cell surface protein further comprises the steps of adding magnetic beads containing the cell surface protein into an eluent for incubation, eluting, and eluting the cell surface protein from the magnetic beads. According to a specific embodiment of the invention, the method for separating and purifying the cell surface protein further comprises the step of carrying out enzymolysis on the protein separated and enriched from the cells to obtain the peptide fragment of the cell surface protein. 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 are 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, the azide preferably includes, but is not limited to, succinimide-tetra-polyethylene glycol-azide (NHS-PEG 4-Azide). 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 at a temperature of 4℃for a period of 1 hour. In the above separat