Mass spectrometry method for detecting intracellular coagulation factor x and application thereof

Abstract

The application belongs to the technical field of mass spectrometry, and particularly relates to a mass spectrometry detection method and application of intracellular blood coagulation factor X, which comprises the following steps: specifically enriching ectopic synthesis of blood coagulation factor X in tumor cells to obtain a protein sample; performing protein sample fixation and treatment on the protein sample by using SDS-PAGE gel electrophoresis, placing the gel in an ice box after scanning the gel by using a gel imaging system, and storing the gel; performing in-gel enzymatic digestion on the blood coagulation factor X in the gel to obtain a peptide segment solution; detecting the endogenous synthesis of blood coagulation factor X in tumor cells by using mass spectrometry technology, and obtaining mass spectrometry data. The application firstly adopts the mass spectrometry technology to detect the blood coagulation factor X synthesized by tumor cells outside normal liver cells, specifically enriches the ectopic synthesis of blood coagulation factor X in glioma cells by using the protein immunoprecipitation technology, performs SDS-PAGE gel electrophoresis, performs in-gel enzymatic digestion, and detects the endogenous synthesis of blood coagulation factor X in tumor cells by using the mass spectrometry technology.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of mass spectrometry, and particularly relates to a mass spectrometry detection method for intracellular blood coagulation factor X and application thereof. BACKGROUND

[0002] Chronic hypercoagulable state in cancer patients can increase the probability of thrombotic events. The risk of venous thromboembolism (VTE) in patients with glioma, lung cancer, colorectal cancer, breast cancer, ovarian cancer and pancreatic cancer can even reach 30%. Although various factors can induce coagulation disorders, FⅩ, as the first protein in the common pathway of coagulation, plays a core role in activating the coagulation cascade.

[0003] The liver has long been considered the only organ for FⅩ synthesis, and there is currently evidence that macrophages, glial cells and other normal somatic cells outside the liver can synthesize FⅩ. However, due to the fact that the half-life of FⅩ is only a few tens of hours, and its detection is easily affected by FⅩ in the blood, traditional Western Blotting, flow cytometry and other technologies based on antigen-antibody detection principle for detecting intracellular proteins are easily disturbed by the above factors and the like. However, there is currently no report on the use of mass spectrometry to detect FⅩ synthesized by normal liver cells and tumor cells. SUMMARY

[0004] To overcome the problems in the related art, the application discloses a mass spectrometry detection method for intracellular blood coagulation factor X and application thereof.

[0005] The technical solution is as follows: a mass spectrometry detection method for intracellular blood coagulation factor X, comprising the following steps:

[0006] S1: specifically enriching blood coagulation factor X synthesized ectopically in tumor cells to obtain a protein sample, i.e., a protein solution;

[0007] S2: detecting the sample by using SDS-PAGE gel electrophoresis for protein sample fixation and treatment, scanning the gel by using a gel imaging system, and placing the gel in an ice box for storage;

[0008] S3: performing in-gel enzymatic digestion on the blood coagulation factor X in the gel to obtain a peptide solution (digested peptides);

[0009] S4: detecting the blood coagulation factor X endogenously synthesized in the tumor cells by using mass spectrometry technology to obtain mass spectrometry data.

[0010] Preferably, the tumor cells are glioma cells, gastric cancer cells, human lung cancer cells or pancreatic cancer cells, etc.

[0011] Further preferably, in step S1, the coagulation factor X ectopically synthesized in the glioma cells is specifically enriched by protein immunoprecipitation technology.

[0012] Further preferably, in step S1, the method for enriching the coagulation factor X ectopically synthesized in the tumor cells comprises the following steps:

[0013] S101: configure a high-efficiency protein lysis solution and place it on ice;

[0014] S102: after washing the cells with PBS, add 300-500ul of high-efficiency protein lysis solution and blow it with a pipette gun to completely cover the cell surface, and lyse on ice for 30min;

[0015] S103: use a cell scraper to scrape off the cells and high-efficiency protein lysis solution and suck them into a clean EP tube, centrifuge at 4°C 12000rpm for 10min, then suck 30-50ul, preferably 50ul of protein supernatant as input group, and the rest of the supernatant is sucked into another EP tube and diluted with high-efficiency protein lysis solution to 1000ul;

[0016] S104: vortex the Dynabeads for 30s, and respectively suck 50ul of Dynabeads into the EP tubes of the experimental group (IP group) and the control group (IgG group), place them on the magnetic stand, and suck off the supernatant, then remove the EP tubes;

[0017] S105: antibody binding: resuspend the Dynabeads with 200ul of Ab Binding and Washing buffer, add 0.75-1ul of goat anti-human Anti-FX antibody / IgG to the experimental group / control group, incubate at 25°C for 10min, discard the supernatant, then add antibody washing solution and discard the supernatant;

[0018] S106: add 100-500ul of high-efficiency protein lysis solution to resuspend the Dynabeads in the experimental group and the control group, seal the EP tube with sealing film, and incubate on a rotary shaker at room temperature for 10min-2h, then suck off the supernatant after the incubation is completed;

[0019] S107: after washing with 200ul of Washing Buffer for 3 times, resuspend the Dynabeads with 100ul of Washing Buffer and suck them into a new EP tube;

[0020] S108: After the supernatant is sucked, 20ul Elution Buffer is added to resuspend the Dynabeads, 5-7ul loading buffer (main component is bromophenol blue which can be used as an indicator) is added, and the EP tube is placed in a magnetic stand. The supernatant is sucked into a clean EP tube for standby, and a protein solution to be detected is obtained.

[0021] Further preferably, the SDS-PAGE gel electrophoresis in step S2 uses a 10% separating gel.

[0022] Further preferably, the SDS-PAGE gel electrophoresis in step S2 includes the following steps:

[0023] S201: Fix the glass plate in the electrophoresis device, with the thin plate facing the inner side, and slowly add the electrophoresis liquid into the tank to cover the inner gel plate;

[0024] S202: Pull out the comb with both hands at the same time, first add 3-5ul, preferably 3ul, of protein marker into the sample wells on both sides, then add 20-25ul of completely dissolved human recombinant FX (rFX) protein into lane 1 as a positive control, add 20-25ul of glioma U87 cell lysate input histone sample into lane 2, add 20-25ul of glioma U87 cell lysate IgG histone sample into lane 3, add 20-25ul of glioma U87 cell lysate IP histone sample into lane 4, add 20-25ul of glioma U87 cell culture supernatant input histone sample into lane 5, add 20-25ul of glioma U87 cell culture supernatant IgG histone sample into lane 6, and add 20-25ul of glioma U87 cell culture supernatant IP histone sample into lane 7;

[0025] S203: After covering the electrophoresis cover, check the electrophoresis device, and the protein electrophoresis is divided into two stages, the first stage is 80V for 30min, and the second stage is 120V for 60min. The generation of bubbles at the metal wire indicates the successful start of electrophoresis.

[0026] In step S2, when the sample is fixed by SDS-PAGE gel electrophoresis, the bromophenol blue is run to the lower part of the gel, and the gel is immediately transferred to the fast coomassie brilliant blue staining solution. First, stain overnight, then wash with double distilled water for 5-15min, preferably 5min, then replace the double distilled water, scan the gel using a gel imaging system, and then store the gel in an ice box.

[0027] In step S3, the coagulation factor X in the gel is subjected to in-gel trypsin digestion, which includes the following steps:

[0028] S301: Cut the gel into small pieces and decolorize using decolorizing solution at 37°C;

[0029] S302: Dehydrate the gel pieces twice with 100% ACN, then reduce with 25 mM dithiothreitol (DTT) at 55°C for 45 minutes and alkylate with 55 mM iodoacetamide in the dark for 20 minutes;

[0030] S303: After removing the reagents, dry the gel pieces completely and digest overnight at 37°C using 0.2 pg trypsin in 50-100 pl, preferably 50 pl digestion buffer;

[0031] S304: Stop the reaction by adding 5 pl 10% formic acid (FA);

[0032] S305: After aspirating the reaction solution, extract the gel pieces twice with 50% ACN / 0.1% FA, then combine and concentrate the centrifugation extract solution and reaction solution to dryness;

[0033] S306: After dissolving the sample in 25 ul 0.1% FA, collect the digestion buffer (peptide fraction solution) for protein identification.

[0034] Further preferably, in step S4, the digested peptide fraction is subjected to sample analysis by an Orbitrap Fusion Lumos mass spectrometer coupled with an Easy-nLC 1200 system, the information of the peptide fraction is obtained by secondary mass spectrometry, and the tumor cell endogenously synthesized blood coagulation factor X is determined through database searching.

[0035] Further preferably, the analysis conditions in step S4 are as follows:

[0036] The peptide fraction is loaded onto a self-packed C18 trapping column of 150 pm x 2 cm and separated on a self-packed C18 analysis column of 150 pm x 30 cm;

[0037] The mobile phase A consists of 0.1% formic acid, and the mobile phase B consists of 80% acetonitrile and 0.1% formic acid;

[0038] The gradient is from 8% to 12% B in 10 minutes, from 12% to 27% in 69 minutes, from 27% to 45% in 28 minutes, from 45% to 95% in 3 minutes, and reaches 95% in 10 minutes;

[0039] The mass spectrometer has a primary mass spectrometry scanning range of 350-1800 m / z, a resolution of 120000, a collision energy (HCD) of 30%, and a secondary mass spectrometry MS2 scan.

[0040] The second inventive purpose of the present application is to provide the use of intracellular blood coagulation factor X as a biomarker for preparing a product for detecting or diagnosing malignant tumors and hypercoagulable state thereof, wherein the biomarker is ectopically synthesized blood coagulation factor X in tumor cells.

[0041] In combination with all the above technical solutions, the present application has the following advantages and positive effects:

[0042] First, in view of the technical problems existing in the prior art and the difficulty in solving the problems, the present application has the following technical effects and advantages:

[0043] The present application first uses mass spectrometry to detect FⅩ synthesized by tumor cells other than normal liver cells, uses protein immunoprecipitation technology to specifically enrich FⅩ ectopically synthesized in glioma cells and perform SDS-PAGE gel electrophoresis, and then uses mass spectrometry to detect FⅩ endogenously synthesized by tumor cells after in-gel digestion.

[0044] The present application has the following advantages:

[0045] High accuracy and sensitivity: the present application uses mass spectrometry to detect FⅩ synthesized by tumor cells other than normal liver cells, which has higher sensitivity than traditional protein detection technology and can more accurately perform amino acid alignment with FⅩ protein sequence.

[0046] Low interference: the present application significantly reduces the interference of FⅩ combined with cell membrane protein receptors and FⅩ in blood.

[0047] Improved time limit: the present application uses mass spectrometry to detect FⅩ synthesized by tumor cells, which can overcome the characteristics of short half-life and difficulty in storage of FⅩ, and significantly improve the time limit for FⅩ detection.

[0048] Second, from the perspective of the product as a whole, the technical solution to be protected by the present application has the following technical effects and advantages:

[0049] The risk of occurrence of VTE is increased by 7 times on average in patients with malignant tumors, and FⅩ synthesized by solid tumor cells (such as glioma) is one of important mechanisms for cancer patients to occur coagulation disorders. FⅩ is an important treatment target for traditional anticoagulation and antithrombotic therapy. The application provides a mass spectrometry detection method for ectopic synthesis of FⅩ in tumor cells, uses high-efficiency, accurate and high-sensitivity mass spectrometry technology to detect endogenous FⅩ synthesized in tumor cells, provides a biomarker for clinical diagnosis of malignant tumors and hypercoagulable states, and provides a new angle and thought for anticoagulant therapy of tumor patients, so that the application can be widely applied in clinical detection.

[0050] Therefore, the application is a very promising detection method, and the application can be widely applied in the diagnosis and research of malignant tumors and hypercoagulable states.

[0051] Thirdly, the creativity of the application as the claim is also embodied in the following important aspects:

[0052] (1) The expected income and commercial value of the technical scheme of the application after transformation are: the technical scheme of the application can widen the screening means of tumor hypercoagulable state after transformation, and add an inspection scheme depending on mass spectrometry technology.

[0053] (2) The technical scheme of the application fills the technical gap in the industry at home and abroad: the technical scheme of the application first proposes to detect blood coagulation factor Ⅹ in extrahepatic tumor cells by using mass spectrometry technology, and provides a new scheme for detecting blood coagulation factor Ⅹ synthesized in cells.

[0054] (3) Whether the technical scheme of the application solves the technical problems that people have been eager to solve but have failed to succeed: compared with the traditional method based on antigen-antibody detection in the past, the technical scheme has extremely high specificity and sensitivity, and is not interfered by FⅩ in blood, and overcomes the shortcomings that FⅩ has a short half-life and is not easy to store and detect.

[0055] (4) Whether the technical scheme of the application overcomes technical bias: the technical scheme overcomes the technical bias of laboratory detection of FⅩ, and in addition to traditional detection methods such as chromogenic substrate, ELISA, and plasma blood coagulation factor Ⅹ activity detection, FⅩ can also be detected based on mass spectrometry technology. BRIEF DESCRIPTION OF DRAWINGS

[0056] The drawings incorporated into the specification and forming part of the specification, show embodiments consistent with the present disclosure, and together with the specification, serve to explain the principles of the present disclosure;

[0057] Figure 1 is an electrophoresis map of SDS-PAGE electrophoresis;

[0058] Figure 2 is a matching result map of mass spectrometry detection of FⅩ peptide segments in tumor cells;

[0059] Figure 3 is a flow chart of the method for detecting coagulation factor X in cells according to the present application;

[0060] Figure 4 is a flow chart of the method for enriching ectopically synthesized coagulation factor X in tumor cells according to the present application;

[0061] Figure 5 is a flow chart of the SDS-PAGE gel electrophoresis according to the present application;

[0062] Figure 6 is a flow chart of the in-gel trypsin digestion of coagulation factor X in the gel according to the present application. DETAILED DESCRIPTION

[0063] In order to make the above objectives, features and advantages of the present application more apparent, specific embodiments of the present application are described in detail below with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in a number of different ways beyond the specific details disclosed herein. It is therefore to be understood that the present application is not limited to the specific embodiments disclosed herein and that modifications can be made in form and detail thereof without departing from the scope of the present application.

[0064] I. Sources of reagents

[0065] Sheep anti-human FX antibody (PAHFX-S; Haematologic Technologies), RIPA lysis buffer (R0010; Solarbio), Protein G magnetic beads (10007D; Solarbio), Coomassie brilliant blue staining solution (G4540; Solarbio), Trypsin (V5117; Promega Madison).

[0066] II. Explanation of the embodiments

[0067] A non-diagnostic method for detecting coagulation factor X (F X) in cells, comprising the following steps:

[0068] S1: specifically enriching ectopically synthesized coagulation factor X in tumor cells to obtain a protein sample;

[0069] S2: using SDS-PAGE gel electrophoresis to fix and process the protein sample, and then placing the gel in an ice box after scanning the gel using a gel imaging system;

[0070] S3: performing in-gel enzymatic digestion of coagulation factor X in the gel to obtain a peptide solution;

[0071] S4: The peptide solution is used to detect endogenous synthesis of blood coagulation factor X in tumor cells by mass spectrometry to obtain mass spectrometry data.

[0072] In one embodiment, the tumor cells are glioma cells, gastric cancer cells, lung cancer cells, or pancreatic cancer cells, etc. The following embodiments take glioblastoma U87 cells as an example.

[0073] Protein immunoprecipitation (IP)

[0074] 1) Configure the high-efficiency protein lysate for protein immunoprecipitation and place it on ice.

[0075] 2) After washing the cells with PBS, add 300-500ul of high-efficiency protein lysate and blow it with a pipette gun to completely cover the cell surface, and lyse on ice for 30min.

[0076] 3) Scrape the cells and lysate with a cell scraper and suck into a clean EP tube. After centrifugation at 4°C 12000rpm for 10min, 50ul of protein supernatant is taken as the input group, and the rest of the supernatant (note that do not suck into the precipitate) is sucked into another EP tube and diluted to 1000ul with high-efficiency protein lysate.

[0077] 4) Vortex for 30s, take 50ul Dynabeads to a 2mL EP tube, place it on a magnetic stand, remove the supernatant, and move the EP tube down.

[0078] 5) Antibody binding: 50ul Dynabeads can bind 12ug of antibody. Add 0.75ul of goat anti-human Anti-FX antibody to the experimental group 200ul Ab Binding and Washing buffer, and incubate at 25°C for 10 minutes. After discarding the supernatant, wash with antibody washing solution and discard the supernatant.

[0079] 6) Add 500ul of high-efficiency protein lysate to resuspend the Dynabeads in the experimental and control groups, seal the EP tube with a sealing film, and incubate on a rotary shaker at room temperature for 2h. After the incubation is completed, the supernatant is removed.

[0080] 7) Wash with 200ul Washing Buffer for 3 times, then add 100ul Washing Buffer to resuspend the Dynabeads and suck them into a new EP tube.

[0081] 8) After removing the supernatant, add 20ul Elution Buffer to resuspend the Dynabeads, then add 7ul loadingbuffer, and boil at 100°C for 10min. Place the EP tube on a magnetic stand, and suck the supernatant into a clean EP tube for standby, to obtain the protein sample.

[0082] SDS-PAGE gel electrophoresis

[0083] 1) Preparation: Select 1.5 mm Bio-rad thick and thin plates, and carefully fix both plates tightly on the gel casting stand. Based on the molecular weight of the target protein detected by mass spectrometry, which is concentrated at 55 kDa, a 10% separating gel was selected for this experiment. After the gel is prepared, it can be used immediately or stored in double-distilled water at 4°C. Prepare the electrophoresis buffer and transfer buffer required for the experiment according to Table 1.

[0084] Table 1. Composition of electrophoresis buffer and transfer buffer

[0085]

[0086] 2) Electrophoresis: Fix the glass plate in the electrophoresis apparatus with the thin plate facing inward, and slowly add the electrophoresis solution into the inner tank until it covers the inner layer of the gel plate. Pull the comb out simultaneously and evenly with both hands. First, add 3 μL of protein marker to both sample wells. Then, add 20-25 μL of completely dissolved recombinant human FX (rFX) protein as a positive control to lane 1. Add 20-25 μL of glioblastoma U87 cell lysate as histone sample to lane 2. Add 20-25 μL of glioblastoma U87 cell lysate as histone sample to lane 3. Add 20-25 μL of glioblastoma U87 cell lysate as histone sample to lane 4. Add 20-25 μL of glioblastoma U87 cell culture supernatant as histone sample to lane 5. Add 20-25 μL of glioblastoma U87 cell culture supernatant as histone sample to lane 6. Add 20-25 μL of glioblastoma U87 cell culture supernatant as histone sample to lane 7. After covering the electrophoresis apparatus with the cap, check the electrophoresis device. Protein electrophoresis is divided into two stages: the first stage is 80V for 30 minutes, and the second stage is 120V for 60 minutes. The electrophoresis is successful when bubbles are generated at the metal wire.

[0087] like Figure 1 As shown, Figure 1 GBM-derived FX were identified by SDS-PAGE from rFX and U87 cell lysates and culture supernatants. Molecular mass marker (M) sizes are in kilodaltons. Gels were stained with Coomassie stain. Among them:

[0088] Lane 1, completely dissolved human recombinant FX (rFX) protein;

[0089] Lane 2, glioblastoma U87 cell lysate input histone sample;

[0090] Lane 3, IgG histone sample from lysate of glioblastoma U87 cells;

[0091] Lane 4, IP fraction of FX from U87 glioblastoma cell lysate;

[0092] Lane 5, input fraction of FX from U87 glioblastoma cell culture supernatant;

[0093] Lane 6, IgG fraction of FX from U87 glioblastoma cell culture supernatant;

[0094] Lane 7, IP fraction of FX from U87 glioblastoma cell culture supernatant.

[0095] Figure 2 FX amino acid sequences obtained from U87 cell lysate and culture supernatant, the matched peptide segments are marked with gray underlined. Among them:

[0096] (a) fully dissolved human recombinant FX (rFX) protein;

[0097] (b) FX from IP fraction of U87 glioblastoma cell lysate;

[0098] (c) FX from IP fraction of U87 glioblastoma cell culture supernatant.

[0099] The above experiments show that the protein immunoprecipitation technique specifically enriches the ectopic synthesis of FⅩ in U87 cell lysate and culture supernatant, providing a new biomarker for clinical diagnosis of malignant tumors and hypercoagulable state, and providing a new angle and idea for anticoagulant therapy for tumor patients.

[0100] Protein spectrum

[0101] When the protein sample obtained by protein immunoprecipitation (IP) is subjected to SDS-PAGE gel electrophoresis using the above steps, when the bromophenol blue runs to the lower part of the gel, the gel is immediately transferred to the fast coomassie brilliant blue staining solution, first stained at room temperature for 2h, then transferred to the 4℃ refrigerator for overnight staining. After completing the staining, wash with double distilled water for 5min, replace with double distilled water, scan the gel using a gel imaging system, and then place the gel in an ice box for storage.

[0102] 1) In-gel FX is subjected to in-gel trypsin digestion.

[0103] Gels were cut into small pieces (1.5 x 1.5 mm) and destained for 40 min at 37°C using destaining solution (50 mM NH4HCO3 and acetonitrile (ACN) 1 : 1 mix). Gel pieces were dehydrated twice with 100% ACN, then reduced with 25 mM DTT for 45 min at 55°C and alkylated with 55 mM iodoacetamide for 20 min in the dark. After removal of reagents, gel pieces were dried completely and digested overnight at 37°C using 0.2 pg trypsin in 50 pl digestion buffer. The reaction was stopped by adding 5 pl 10% formic acid (FA). After aspiration of the reaction solution, gel pieces were extracted twice with 50% ACN / 0.1% FA, the centrifugation extracts and reaction solution were combined and concentrated to dryness. After dissolving the sample in 25 ul 0.1% FA, the digestion buffer was collected for protein identification.

[0104] 2) The digested peptides were analyzed by an Orbitrap Fusion Lumos mass spectrometer coupled to an Easy-nLC 1200 system (Thermo Fisher Scientific).

[0105] Peptides were loaded onto a 150 pm x 2 cm self-packing C18 trap column (particle size 3 pm, Dr. MASCH GmbH, Germany) and separated on a 150 pm x 30 cm self-packing C18 analytical column (particle size 1.9 pm, Dr. MASCH GmbH).

[0106] The mobile phase A consisted of 0.1% formic acid and the mobile phase B consisted of 80% acetonitrile and 0.1% formic acid.

[0107] The gradient was from 8% to 12% B in 10 min, from 12% to 27% in 69 min, from 27% to 45% in 28 min, from 45% to 95% in 3 min, reaching 95% in 10 min,

[0108] The primary mass spectrometry scan range in the Orbitrap was 350-1800 m / z with a resolution of 120000, collision energy (HCD) of 30% and secondary mass spectrometry MS2 scan with a resolution of 30000.

[0109] The LC-MS / MS runs were searched against the Swiss Human Protein Database (uniprot, release date 29 September 2020) containing 20311 sequence entries using Proteome Discoverer (version 2.2) to align the primary and secondary spectra of the LC-MS / MS runs with the database.

[0110] Search criteria are as follows: trypsin enzyme cleavage site K and R; allow twice missed cleavage; carbamoylmethylation (C) is set as a fixed modification; oxidation (M) is set as a dynamic modification; all primary mother ions collected in the Orbitrap mass analyzer allow a maximum mass error of 20 ppm; the secondary fragment ion mass error threshold is 0.02 Da. The matched peptides are obtained using a high confidence score filter (FDR < 1%), and the corresponding MS / MS spectra are manually checked to identify the FX ectopically synthesized by glioblastoma through LC / MS-MS.

[0111] Table 2 is the search result of ectopically synthesized FX in rFX, U87 cell lysate IP group histone sample and U87 cell culture supernatant protein sample by mass spectrometry.

[0112] Table 2. Determination of ectopic FX by LC-MS / MS method

[0113]

[0114] Note: coagulation factor X (FX): coagulation factor X; OS = Homo sapiens: Latin name of the species; OX = Organism Taxonomy, species classification database Taxonomy ID; GN = Gene Name, gene name; PE = Protein Existence, reliability of protein; SV = Sequence version, protein sequence version

[0115] The above experiments show that the FX synthesized by U87 cells is successfully detected by using high-efficiency, precise and high-sensitivity mass spectrometry, which overcomes the shortcomings of short half-life and difficulty in storage of FⅩ, and effectively improves the time limit for detecting FX.

[0116] III. Application Examples

[0117] The mass spectrometry method for detecting intracellular coagulation factor X provided in Embodiment 1 of the present application is used to detect FⅩ synthesized by normal liver extracellular tumor cells, and the FⅩ synthesized by the normal liver extracellular tumor cells can be used as a biomarker for detecting or diagnosing malignant tumors and hypercoagulable states.

[0118] It should be noted that the mass spectrometry method for detecting intracellular coagulation factor X provided in the embodiment 1 of the present application is a non-diagnostic purpose in vitro experimental method for detecting intracellular coagulation factor X, which is essentially the protection of detection steps, detection conditions and detection parameters, and the obtained result is to identify the FX synthesized ectopically by glioblastoma, thereby obtaining a high-efficiency, precise and high-sensitivity non-diagnostic purpose in vitro detection method for detecting intracellular coagulation factor X. The above method is not a method for directly identifying, determining or eliminating the cause or lesion, and the result value obtained by the disclosed mass spectrometry method is not in the form of final diagnosis, and the diagnostic result or health status of the same subject cannot be known.

[0119] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement made by any person skilled in the art within the technical range disclosed by the present application, as long as it is within the spirit and principle of the present application, should be covered within the protection scope of the present application.

Claims

1. A method for the mass spectrometric detection of intracellular factor Xa, characterized in that: The method comprises the following steps: S1: specifically enriching the ectopic synthesis of blood coagulation factor X in tumor cells to obtain a protein sample; The method for enriching the ectopic synthesis of blood coagulation factor X in tumor cells comprises the following steps: S101: configure a high-efficiency protein lysate and place it on ice; S102: after washing the cells with PBS, add a corresponding amount of high-efficiency protein lysate and blow it with a pipette gun to completely cover the cell surface, and lyse for 30 min on ice; S103: scrape the cells and lysate with a cell scraper and suck them into a clean EP tube, centrifuge at 12000 rpm for 10 min at 4 DEG C, and then suck 30-50 ul of the protein supernatant as the input group, and the remaining supernatant is sucked into another EP tube and diluted with high-efficiency protein lysate to 1000 ul; S104: vortex for 30 s, suck 50 ul of Dynabeads into a 2 ml EP tube, place it on a magnetic stand, and suck the supernatant, and then remove the EP tube; S105: antibody binding: resuspend the Dynabeads with 200 ul of AbBinding and Washing buffer, add 0.75-1 ul of goat anti-human Anti-FX antibody / IgG to the experimental group / control group respectively, incubate at 25 DEG C for 10 min, discard the supernatant, and then add antibody washing solution and discard the supernatant; S106: add 100-500 ul of sample lysate to resuspend the Dynabeads in the experimental group and the control group, seal the EP tube with a sealing film, and incubate on a rotary shaker at room temperature for 10 min-2 h, and then suck the supernatant after the incubation is completed; S107: wash with 200 ul of Washing Buffer for 3 times, resuspend the Dynabeads with 100 ul of Washing Buffer and suck them into a new EP tube; S108: place the EP tube on a magnetic stand, suck the supernatant, resuspend the Dynabeads with 20 ul of Elution Buffer, add 5-7 ul of loading buffer, and boil at 100 DEG C for 10 min, place the EP tube on a magnetic stand, suck the supernatant into a clean EP tube for standby, and obtain the protein sample; S2: use SDS-PAGE gel electrophoresis to fix and process the protein sample, scan the gel by using a gel imaging system, and then place the gel in an ice box for storage; S3: perform in-gel enzymatic digestion on the blood coagulation factor X in the gel to obtain a peptide solution; S4: use mass spectrometry to detect the blood coagulation factor X endogenously synthesized in tumor cells to obtain mass spectrometry data. The method can overcome the interference of cell membrane protein receptor-bound blood coagulation factor X and blood coagulation factor X in blood, and detect the blood coagulation factor X endogenously synthesized in normal liver cells outside tumor cells.

2. The method of claim 1, wherein the method is used for detecting Factor Xa in a cell. The tumor cells are glioma cells, gastric cancer cells or pancreatic cancer cells.

3. The method of claim 1, wherein the method is used for detecting Factor Xa in a cell. In step S2, the SDS-PAGE gel electrophoresis selects a separation gel with a concentration of 10%.

4. The method of claim 3, wherein the method is used for detecting Factor Xa in a cell. In step S2, the SDS-PAGE gel electrophoresis comprises the following steps: S201: Fix the glass plate in the electrophoresis device, with the thin plate facing the inner side, slowly add the electrophoresis liquid into the inner tank until it covers the inner layer of the glass plate; S202: Pull out the comb with both hands at the same time and evenly, first add 3-5ul of protein marker in the sample holes on both sides, then add 20-25ul of fully dissolved human recombinant FX protein in lane 1 as a positive control, add 20-25ul of glioma U87 cell lysate input histone sample in lane 2, add 20-25ul of glioma U87 cell lysate IgG histone sample in lane 3, add 20-25ul of glioma U87 cell lysate IP histone sample in lane 4, add 20-25ul of glioma U87 cell culture supernatant input histone sample in lane 5, add 20-25ul of glioma U87 cell culture supernatant IgG histone sample in lane 6, and add 20-25ul of glioma U87 cell culture supernatant IP histone sample in lane 7; S203: After covering the electrophoresis cover, check the electrophoresis device, the protein electrophoresis is divided into two stages, the first stage is 80V for 30min, and the second stage is 120V for 60min, and the generation of bubbles at the metal wire is the start of successful electrophoresis.

5. The method of claim 4, wherein the method is used for the detection of Factor Xa in a cell. In step S2, the detection sample is fixed by SDS-PAGE gel electrophoresis, and when the bromophenol blue runs to the lower part of the gel, the gel is immediately transferred to a fast coomassie brilliant blue staining solution, after staining, the gel is washed with double distilled water for 5-15min, and after scanning the gel by using a gel imaging system, the gel is placed in an ice box for storage.

6. The method of claim 1, wherein the method is used for detecting Factor Xa in a cell. In step S3, the coagulation factor X in the gel is subjected to in-gel trypsin digestion, which specifically includes the following steps: S301: Cut the gel into small pieces and use a decolorizing solution to decolorize at 37°C; S302: Dehydrate the gel pieces with 100% ACN twice, then reduce with 25mM dithiothreitol at 55°C for 45min, and alkylate with 55mM iodoacetamide in the dark for 20min; S303: After removing the reagent, dry the gel pieces completely, and digest overnight at 37°C using 0.2ug trypsin in 50-100ul digestion buffer; S304: Add 5ul of 10% formic acid to stop the reaction; S305: Absorb the reaction liquid into a new EP tube, add extraction liquid to cover the gel particles, extract at 37°C for 30min, absorb the extraction liquid into the EP tube of the reaction liquid, repeat the extraction once, mix the two extraction liquids and the reaction liquid, and concentrate to dryness; S306: After dissolving the sample in 25ul of 0.1% formic acid, collect the digestion buffer for protein identification.

7. The method of claim 1, wherein the method is used for detecting Factor Xa in a cell. In step S4, the peptide solution is analyzed by an Orbitrap Fusion Lumos mass spectrometer coupled with an Easy-nLC1200 system, the information of the peptide is obtained by secondary mass spectrometry, and the coagulation factor X endogenously synthesized in the tumor cells is determined through database search.

8. The method of claim 7, wherein the method is used for detecting Factor Xa in a cell. The analysis conditions in step S4 are: Peptide segments were loaded onto a 150 pm x 2 cm self-packed C18 trapping column and separated on a 150 pm x 30 cm self-packed C18 analytical column; The mobile phase A consisted of 0.1% formic acid and the mobile phase B consisted of 80% acetonitrile and 0.1% formic acid; The gradient was from 8% to 12% B in 10 min, from 12% to 27% in 69 min, from 27% to 45% in 28 min, from 45% to 95% in 3 min, and reached 95% in 10 min; The mass spectrometer had a range of 350-1800 m / z for the first mass spectrum scan with a resolution of 120000 and a collision energy (HCD) of 30% for the second mass spectrum MS2 scan.

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