Method for extracting perfluorooctane sulfonic acid combined with receptor in cells based on membrane receptor

Abstract

The invention discloses a G protein coupled receptor family membrane receptor GPR40 related to eukaryotic expression and glycometabolism effect, and a method for extracting and detecting the concentration of a compound combined with the GPR40 receptor in a cell based on the affinity of receptor protein. The detection method comprises the following steps: eukaryotically expressing a membrane receptor GPR40 protein with a Flag tag in a human embryonic kidney cell HEK293, and after perfluorooctane sulfonic acid (PFOS) is exposed, extracting and purifying a GPR40-PFOS compound in a cell lysis solution by utilizing Protein G magnetic beads and a Flag antibody; after uncombined PFOS is washed away by a cracking buffer solution containing a protease inhibitor, acetonitrile is added, and PFOS in the compound is extracted; and centrifuging and treating with a 0.22 mu m filter membrane, and detecting the concentration of the PFOS by using a high performance liquid chromatography-mass spectrometer (HPLC-MS / MS). The method can directly detect the concentration of PFOS combined with the GPR40 receptor, can be used for screening other perfluorinated compounds and environmental pollutants which potentially act on the GPR40 receptor to play a glycometabolism effect, can simplify the steps of effect-oriented analysis, and plays an important role in compound recognition and screening.

Description

Technical Field

[0001] This invention relates to a method for extracting the concentration of perfluorooctane sulfonic acid bound to the receptor within cells based on membrane receptors, belonging to the field of bioanalytical chemistry. Background Technology

[0002] Perfluorooctane sulfonate (PFOS) is a typical perfluorinated compound widely used in production and daily life due to its hydrophobic and oleophobic properties, such as in textiles, leather, food contact materials, non-stick cookware coatings, and medical products. With the widespread use of these products and the persistent nature of PFOS, environmental pollution from PFOS is becoming increasingly serious. Humans can be exposed to PFOS through food, drinking water, air, and skin, posing a serious threat to human health. PFOS exhibits carcinogenicity, endocrine disruption toxicity, reproductive and developmental toxicity, immunotoxicity, lipid metabolism toxicity, and glucose and lipid metabolism disruption effects. PFOS exerts its toxic effects through receptors, and the exposure concentration determines the magnitude of the toxic effect. Currently, the concentration at which PFOS exerts its toxic effect is determined based on its theoretical exposure concentration, but this method is prone to underestimating its toxicity. Therefore, determining the concentration of PFOS that binds to its receptors (the actual concentration at which it exerts its toxic effect) is crucial for accurately assessing its toxicity.

[0003] GPR40, a member of the G protein-coupled receptor family, is a cell membrane receptor containing seven transmembrane α-helical regions. GPR40 is highly expressed in pancreatic β-cells and is an important mediator of insulin secretion, participating in the synthesis and secretion of insulin and other proteins. PFOS can interfere with insulin secretion by binding to GPR40, leading to glucose metabolism disorders. Therefore, extracting PFOS bound to GPR40 is essential for determining the actual effective concentration of PFOS to exert its toxic effects.

[0004] Protein (receptor) affinity-based compound extraction offers a solution to this problem. Currently, methods based on receptor affinity are mainly applied to environmental samples, such as water, soil, and air samples, and are primarily based on nuclear receptors, obtained from E. coli prokaryotic expression systems. However, prokaryotic expression suffers from drawbacks: expensive purification packing materials, large purification tags that may affect protein function, and unmodified prokaryotic proteins may not possess natural activity, potentially leading to non-specific adsorption. Eukaryotic expression methods, on the other hand, ensure successful protein folding, and receptor binding to magnetic beads guarantees a lower false-positive rate (non-specific adsorption). Human embryonic kidney cells (HEK293) are commonly used eukaryotic expression cells due to their efficient expression of exogenous genes. Furthermore, during contaminant entry into cells, contaminants may act on membrane receptors earlier than nuclear receptors, causing toxic effects. Therefore, constructing a eukaryotic expression system for GPR40 based on HEK293 cells and extracting and purifying the intracellular PFOS concentration bound to GPR40 is of significant importance for the toxicity assessment of PFOS and compounds with similar effects. Summary of the Invention

[0005] The problem this invention aims to solve is how to determine the intracellular concentration of PFOS that binds to the membrane receptor GPR40. This invention involves extracting intracellular PFOS that binds to GPR40 in HEK293 cells overexpressing the membrane receptor GPR40, based on protein (receptor) affinity, and determining the PFOS concentration using HPLC-MS / MS.

[0006] The method provided by this invention includes the following steps:

[0007] (1) A recombinant plasmid capable of expressing the Flag tag GPR40 was obtained by restriction endonuclease and ligase methods. The Flag tag membrane receptor GPR40 was overexpressed in HEK293 cells by plasmid identification, amplification and extraction, and cell transfection.

[0008] (2) HEK293 cells overexpressing GPR40 were exposed to different concentrations of PFOS, and then the cells were lysed. The magnetic bead-GPR40-PFOS complex was extracted by incubation with magnetic beads-antibody, and the complex was washed with buffer to remove unbound PFOS.

[0009] (3) PFOS bound to the receptor was extracted by adding acetonitrile and heating. After filtration through a filter membrane, the concentration of PFOS bound to GPR40 was detected by HPLC-MS / MS.

[0010] As a preferred method, the amount of GPR40 recombinant plasmid transfected into HEK293 cells was 30 μg, the transfection reagent was Turbofect, and the transfection time was 24 hours.

[0011] As preferred, the concentrations of PFOS were 25 μM and 50 μM, with 0.1% DMSO as the control group, and the exposure time was 48 hours.

[0012] Preferably, the cell lysis buffer is a 1% NP40 solution containing a protease inhibitor, the magnetic beads are 20 μL of Protein G magnetic beads, the antibody is 1 μL of Anti-Flag mouse anti-antibody, the incubation time is 12 hours, and the incubation temperature is 4°C.

[0013] Preferably, the buffer used to wash the GPR40-PFOS complex is a 1% NP40 solution containing a protease inhibitor, and the washing is performed 3 times for 5 minutes each time.

[0014] As a preferred option, the supernatant from the third washing is retained for subsequent testing to exclude the influence of unbound PFOS.

[0015] Preferably, the volume of acetonitrile used to extract PFOS is 500 μL, the heating temperature is 80 °C, the time is 10 minutes, and then the mixture is centrifuged at 12000 rpm for 10 minutes, and the supernatant is collected.

[0016] As a preferred method, the sample was filtered through a 0.22 μm filter membrane and then analyzed by HPLC-MS / MS with a sample loading volume of 5 μL.

[0017] As a preferred method, a standard curve is plotted based on PFOS standards, and the concentration of PFOS bound to GPR40 is calculated.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) This invention provides a method for extracting PFOS bound to the GPR40 membrane receptor from cells. By constructing a HEK293 eukaryotic cell line overexpressing GPR40, and using Protein G-coated magnetic beads and Flag-tagged antibodies, PFOS bound to the GPR40 membrane receptor is extracted from cells through incubation with cell lysis buffer. The concentration of PFOS is then detected using HPLC-MS / MS. The method developed in this invention can determine the concentration of PFOS directly bound to the intracellular receptor, i.e., the concentration that actually exerts its effect, providing a more accurate method for the toxicological assessment of PFOS.

[0020] (2) The method for extracting PFOS based on receptor affinity proposed in this invention can be used to detect different biological effects and types of pollutants by changing different receptors (membrane receptors, nuclear receptors, etc.), thereby expanding the scope of this detection technology and its application. Attached Figure Description

[0021] Figure 1The protein sequence and coding sequence of the GPR40 receptor.

[0022] Figure 2 HEK293 cells were transfected with GPR40 recombinant plasmid or empty vector plasmid for 24 hours. After enrichment with magnetic beads, GPR40 expression was detected by anti-Flag antibody.

[0023] Figure 3 After exposing HEK293 cells transfected with the GPR40 membrane receptor to 25 μM and 50 μM for 48 hours, the concentration of PFOS bound to GPR40 in the cells was detected using the present invention. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the content described.

[0025] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1: A method for extracting PFOS that binds to the receptor within cells based on the membrane receptor GPR40, the specific steps of which are as follows:

[0028] Construction of GPR40 recombinant plasmid:

[0029] The human GPR40 sequence number obtained from NCBI is NM_005303, such as... Figure 1 As shown, the protein and coding sequences of the GPR40 receptor were successfully located. XhoI / KpnI was added to the 5' end of the target gene primer, and the target gene fragment was prepared by PCR amplification. The vector GV219 (cloning site XhoI / KpnI) was selected, and the PCR product was ligated to the vector using restriction endonucleases (XhoI / KpnI) and T4 ligase. The ligation product was transformed into *E. coli*, plated, and single colonies were picked for PCR identification to confirm positive clones. After plasmid construction, plasmid amplification and extraction were performed. Quantification and quality assessment of the recombinant plasmid were conducted using a micro-ultraviolet spectrophotometer, and the plasmid was stored at -20℃.

[0030] Constructing a HEK293 cell model overexpressing the GPR40 receptor:

[0031] When the HEK293 cell density in the T75 cell flask reached 80%, 30 μg of the GPR40 recombinant plasmid was transfected into HEK293 cells according to the Invitrogen Turbofect transfection kit. After 24 hours of transfection, GPR40 protein expression was achieved. GPR40 protein was extracted and purified with or without Protein G magnetic beads, and the samples were analyzed by Western blot. Figure 2 As shown, in HEK293 cells overexpressing the GPR40 receptor, GPR40-Flag expression was detectable in the cell lysate without Protein G magnetic beads for GPR40 enrichment; after enrichment with magnetic beads, the GPR40-Flag content was even higher; after lysis of HEK293 cells transfected with an empty vector plasmid, GPR40-Flag protein was not detected regardless of whether magnetic beads were used for enrichment. This indicates that HEK293 cells overexpressing the GPR40 receptor were successfully constructed, and that magnetic beads can effectively enrich the GPR40 receptor protein.

[0032] Extraction of the GPR40-PFOS complex:

[0033] (1) After transfecting HEK293 cells with GPR40 recombinant plasmid for 24 hours, the cells were exposed to 0.1% DMSO (control group), 25 μM and 50 μM PFOS for 48 hours; then 2.5 mL of 1% NP40 lysis buffer (containing protease inhibitor) was added and the cells were lysed on ice for 30 minutes.

[0034] (2) Use a cell scraper to collect cells into a 5 mL sterile centrifuge tube and continue to lyse for 30 minutes. Invert the centrifuge tube every 6 minutes and then centrifuge at 12000 rpm and 4℃ for 15 minutes. Collect the supernatant into a new 5 mL sterile centrifuge tube.

[0035] (3) During cell lysis, 20 μL of Protein G magnetic beads and 1 μg of Anti-flag antibody were co-incubated with 200 μL of 1% NP40 lysis buffer containing protease inhibitor at 4°C for 1 hour to allow the antibody to bind to the magnetic beads. The magnetic bead-antibody complex was adsorbed using a magnetic rack, and the supernatant was discarded.

[0036] (4) Incubate the protein in (2) and the magnetic bead-antibody complex in (3) at 4°C overnight (on a rotary mixer), and use a magnetic rack to adsorb the magnetic bead-antibody-GPR40-PFOS complex, and discard the supernatant.

[0037] (5) The complex in (4) was washed with 500 μL of 1% NP40 lysis buffer containing protease inhibitor (on a rotary mixer) for 5 minutes each time, for 3 times. The magnetic bead-antibody-GPR40-PFOS complex was obtained using a magnetic rack, and the supernatant after the third wash was collected for HPLC-MS / MS detection to exclude the influence of unbound PFOS.

[0038] Acetonitrile extraction of PFOS from the GPR40-PFOS complex:

[0039] Add 500 μL of acetonitrile to the complex obtained in step (5), heat at 80°C for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and extract PFOS from the magnetic bead-antibody-GPR40-PFOS complex; at the same time, take 250 μL of the supernatant from the third wash in (5), add 500 μL of acetonitrile, heat at 80°C for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and extract PFOS.

[0040] HPLC-MS / MS detection of PFOS:

[0041] The acetonitrile-extracted solution from the previous step was filtered through a 0.22 μm filter membrane and transferred into a sample vial for routine HPLC-MS / MS analysis. The PFOS concentrations for the standard curve were 0.1 ppb, 1 ppb, 5 ppb, 10 ppb, 50 ppb, 0.1 ppb, 100 ppb, and 200 ppb. The PFOS concentration in the sample was calculated based on the standard curve. The results are as follows... Figure 3 As shown, PFOS was not detected in the control group (0.01% DMSO), the intracellular PFOS concentration binding to the GPR40 receptor in the 25 μM PFOS exposure group was 0.89 ppb, and the intracellular PFOS concentration binding to the GPR40 receptor in the 50 μM PFOS exposure group was 1.30 ppb. PFOS was not detected in the supernatant after the third wash of the magnetic bead-antibody-GPR40-PFOS complex. These findings demonstrate that the method constructed in this invention can be used to detect intracellular PFOS that directly binds to the receptor.

Claims

1. A method for extracting intracellular PFOS concentrations bound to receptors based on membrane receptors, characterized in that: The eukaryotic membrane receptor GPR40 was expressed, and the intracellular concentration of PFOS binding to the GPR40 receptor (the concentration that actually exerts the biological effect) was extracted and detected. The main steps included: (1) Overexpression of the Flag-tagged membrane receptor GPR40 in HEK293 cells; (2) After exposing cells in (1) to different concentrations of PFOS, the GPR40-PFOS complex was extracted by magnetic bead method. (3) The concentration of PFOS bound to GPR40 was detected by high performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

2. The method for extracting intracellular PFOS concentration bound to the receptor based on membrane receptors according to claim 1, characterized in that: The full coding sequence (CDS) of human GPR40 was found in NCBI. A Flag tag sequence was added to the C-terminus, and then cloned into the multiple cloning site of a eukaryotic expression vector to construct the GPR40-Flag recombinant plasmid. The GPR40-Flag recombinant plasmid was transiently transfected into HEK293 cells to express the GPR40-Flag protein.

3. The method for extracting intracellular PFOS concentration bound to the receptor based on membrane receptors according to claim 1, characterized in that: HEK293 cells overexpressing GPR40 were exposed to different concentrations of PFOS for 48 hours, and then the GPR40-PFOS complex was extracted using the magnetic bead method.

4. The method for extracting intracellular PFOS concentration bound to the receptor based on membrane receptors according to claim 1, characterized in that: The GPR40-PFOS complex was washed with buffer to remove unbound PFOS, and the PFOS in the GPR40-PFOS complex was extracted with acetonitrile before the PFOS concentration was detected by HPLC-MS / MS.

5. A method for extracting intracellular PFOS concentrations bound to receptors based on membrane receptors according to claims 1 and 2, characterized in that: The eukaryotic expression vector was GV219, the target sequence was the full coding sequence of GPR40 (NM_005303), and the restriction endonucleases were Xho I and Kpn I. HEK293 cells were cultured in T75 cell culture flasks and transfected with 30 μg of the GPR40 recombinant plasmid.

6. The method for extracting intracellular PFOS concentration bound to the receptor based on membrane receptors according to claims 1 and 3, characterized in that: The GPR40-PFOS complex was extracted using a magnetic bead-Flag antibody immunoassay, with 20 μL of Protein G magnetic beads and 1 μg of Anti-Flag antibody.

7. A method for extracting intracellular PFOS concentrations bound to receptors based on membrane receptors according to claims 1 and 4, characterized in that: The GPR40-PFOS complex was washed three times with 1% NP40 lysis buffer containing protease inhibitors (50 mM Tris, 150 mM NaCl, 1% NP-40) to remove unbound PFOS; during extraction, 500 μL of acetonitrile was added.

8. A method for extracting intracellular PFOS concentration bound to receptors based on membrane receptors according to claims 1 and 7, characterized in that: The supernatant from the third wash was tested on the machine to rule out the influence of unbound PFOS.

9. A method for extracting intracellular PFOS concentration bound to receptors based on membrane receptors according to claims 1-8, characterized in that: The eukaryotic membrane receptor GPR40 was expressed. The GPR40-PFOS complex was extracted from cells using a magnetic bead-Flag antibody immunoassay. After acetonitrile extraction, the concentration of PFOS bound to the GPR40 receptor was detected by HPLC-MS / MS.

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