Cancer cell and fibroblast specific proteome separation method based on FFPE pathological section

Abstract

The invention belongs to the technical field of proteomics, and particularly relates to a cancer cell and fibroblast specific proteome separation method based on an FFPE pathological section. According to the cancer cell and fibroblast specific proteome separation method based on the FFPE pathological section, tumor cells are specifically labeled in an FFPE sample through poly-HRP direct-labeled pan-CK and EpCAM, fibroblast is specifically labeled through poly-HRP direct-labeled aSMA and FAP, and then a biotin magnetic bead enrichment technology is combined, so that simple, efficient, high-specificity and low-cost proteomics analysis can be realized, and the method is suitable for clinical application. The method solves the problems of high sample complexity, complicated operation, high cost, low FFPE sample utilization rate and the like in the prior art, and has important scientific value and clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of proteomics technology, specifically relating to a method for isolating cancer cell and fibroblast-specific proteomes based on FFPE pathological sections. Background Technology

[0002] Currently, tumor proteomics research techniques mainly include: proteomics analysis based on fresh frozen samples, proteomics analysis based on FFPE samples, proteomics analysis after flow cytometry sorting, proteomics analysis based on laser capture microdissection, mass spectrometry imaging, and single-cell proteomics.

[0003] Proteomics analysis based on fresh frozen samples involves rapidly freezing and preserving fresh frozen tissue under liquid nitrogen or ultra-low temperature conditions, followed by extraction of total protein after lysis buffer or ultrasonic disruption. The extracted proteins are then enzymatically digested and identified and quantified using LC-MS / MS. Proteomics analysis based on FFPE samples involves dewaxing the samples, extracting total protein using lysis buffer or high-temperature, high-pressure treatment, followed by enzymatic digestion and analysis using LC-MS / MS.

[0004] Post-flow cytometry sorting proteomics analysis utilizes flow cytometry to separate specific cell populations based on cell surface markers. Sorted cells are then lysed and proteins extracted, followed by analysis using LC-MS / MS. Laser-capture microdissection proteomics analysis uses a laser to precisely dissect target regions (such as tumor cells) in FFPE samples, separating them from complex tissues. Dissected cells are then lysed and proteins extracted, followed by analysis using LC-MS / MS.

[0005] Mass spectrometry imaging involves scanning directly stained tissue sections to analyze the spatial characteristics of the tumor microenvironment and reveal the spatial distribution of proteins. Single-cell proteomics uses microfluidic technology, flow cytometry, or laser capture microdissection to isolate individual cells from complex tissues or mixed samples. After cell lysis, LC-MS / MS is used to study the protein expression profiles of individual cells.

[0006] Proteomics analysis based on fresh frozen samples and FFPE samples yields whole-tissue proteomics. Tumor tissues are composed of multiple cell types, and the complex cellular heterogeneity results in a large number of non-tumor-related signals in the proteomics data, masking the molecular characteristics specific to tumor cells and thus limiting in-depth analysis of tumor-specific proteins. Secondly, due to the varying proportions of tumor cells in the tissue, their specific proteins are often masked by high-abundance proteins or non-tumor cell proteins, further increasing the difficulty of detection. In addition, the acquisition and preservation conditions for fresh frozen samples are demanding, making large-scale application in clinical practice difficult; proteins extracted directly from FFPE samples undergo protein cross-linking due to formalin fixation, preventing proteases from effectively cleaving the cross-linked regions and affecting the coverage of mass spectrometry identification.

[0007] Proteomics analysis following flow cytometry sorting and proteomics analysis following laser capture microdissection are cumbersome, requiring high-precision equipment and specialized technicians; they are also costly and unsuitable for large-scale sample analysis. Furthermore, flow cytometry sorting is only applicable to fresh samples, and laser capture microdissection has limited applicability to FFPE samples, making it unsuitable for large-scale retrospective studies.

[0008] Mass spectrometry imaging technology has low sensitivity, making it difficult to detect low-abundance proteins. Data analysis is complex and requires a high-quality reference spectral library. Furthermore, the equipment is expensive, its current adoption rate is low, and its applicability is limited. Single-cell proteomics is technically challenging, with complex sample preparation and data analysis, resulting in high costs and currently limiting its application to small-scale studies.

[0009] Therefore, there is a need for a proteomics analysis method based on FFPE samples that is simpler to operate, lower in cost, and has higher sample utilization. Summary of the Invention

[0010] The first objective of this invention is to provide a mass spectrometry method for identifying cell-specific proteins based on FFPE sample tissue sections.

[0011] The second objective of this invention is to provide an application of the mass spectrometry identification method of the first aspect of this invention.

[0012] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0013] A first aspect of the present invention provides a mass spectrometry method for identifying cell-specific proteins based on FFPE sample tissue sections, comprising the following steps:

[0014] 1) After dewaxing and rehydration of FFPE sample tissue sections, heat treatment was performed to repair antigens;

[0015] 2) Incubate the samples with a cell-specific antibody labeled with horseradish peroxidase;

[0016] 3) Biotin labeling;

[0017] 4) Prepare protein lysis samples;

[0018] 5) Purification of streptomycin avidin;

[0019] 6) Prepare mass spectrometry samples and perform mass spectrometry identification;

[0020] The cells include tumor cells and fibroblasts.

[0021] In some embodiments of the present invention, the area of ​​the slice in step 1) is 70–200 mm². 2 The thickness is 3-5 μm.

[0022] In some embodiments of the present invention, the heat treatment in step 1) includes: covering the slice with antigen retrieval buffer and then heating it.

[0023] In some embodiments of the present invention, the temperature of the heat treatment is 80 to 120°C.

[0024] In some embodiments of the present invention, the heating treatment time is 10 to 30 minutes.

[0025] In some embodiments of the present invention, the cell-specific antibody in step 2) includes tumor cell-specific antibody and fibroblast-specific antibody;

[0026] In some embodiments of the present invention, the tumor cell-specific antibodies include pan-CK and EpCAM.

[0027] In some embodiments of the present invention, the fibroblast-specific antibody includes aSMA and FAP.

[0028] In some embodiments of the present invention, the horseradish peroxidase in step 2) is a polyhortradish catalase, specifically composed of 3 to 5 identical HRP homopolymers, which can covalently couple with multiple streptavidin molecules simultaneously, significantly amplifying the signal and improving labeling efficiency.

[0029] In some embodiments of the present invention, horseradish catalase is labeled on a specific antibody using ProteinA / G-Poly HRP40.

[0030] In some embodiments of the present invention, the biotin labeling in step 3) is a biotinylated tyrosine amide treatment of the sample;

[0031] In some embodiments of the present invention, the treatment concentration of the biotinylated tyrosine amide is 10–20 μg / mL.

[0032] In some embodiments of the present invention, the treatment time of the biotinylated tyrosine amide is 20 to 30 minutes.

[0033] In some embodiments of the present invention, endogenous peroxidase blocking and occlusion operations are performed before step 3).

[0034] In some embodiments of the present invention, the method for preparing protein lysate samples in step 4) includes: treating the protein with lysate and sonicating.

[0035] In some embodiments of the present invention, the lysis buffer includes Tris-HCl buffer, a reducing agent (e.g., DTT), a metal chelating agent (e.g., EDTA, EGTA), and a protease inhibitor (e.g., PMSF).

[0036] In some embodiments of the present invention, the lysis buffer consists of: 0.1 M Tris-HCl, 0.1 M DTT, 2 mM EDTA, 1 mM PMSF, and pH 8.0.

[0037] In some embodiments of the present invention, ice bath ultrasonic pulverization is used. The conditions for non-contact ultrasonication are: 540W, ultrasonication for 10 seconds, stop for 10 seconds, for a total of 30 cycles. After ultrasonication for 5 minutes, the sample is removed, centrifuged in a handheld centrifuge for 3-5 seconds, and ultrasonication is repeated once.

[0038] In some embodiments of the present invention, the method for purifying streptavidin in step 5) includes: enriching and purifying biotin-labeled proteins using streptavidin magnetic beads.

[0039] In some embodiments of the present invention, the preparation of the mass spectrometry sample in step 6) includes the following steps:

[0040] Reduction, alkylation, enzymatic hydrolysis, desalting, and drying.

[0041] In some embodiments of the present invention, the reduction is performed using DTT treatment at a concentration of 10–30 mM for a duration of 100–150 min and a temperature of 35–40 °C.

[0042] In some embodiments of the present invention, the enzymatic digestion is performed using trypsin, and the mass ratio of trypsin to protein sample is 1:25 to 100.

[0043] In some embodiments of the present invention, steps 1), 2), and 3) are performed in a fully automatic intelligent dyeing machine.

[0044] The beneficial effects of this invention are:

[0045] 1) Fully automated operation: The dewaxing, staining and antigen retrieval steps of FFPE sections are performed in a fully automated intelligent staining machine, which effectively avoids contamination of mass spectrometry detection by human proteins and improves experimental repeatability and result reliability.

[0046] 2) Antigen retrieval optimization: Heat-induced antigen retrieval (HIER) technology significantly removes the effect of formalin fixation on protein cross-linking, restores antigenicity, and provides high-quality proteins for subsequent antibody labeling and mass spectrometry analysis.

[0047] 3) High-specificity labeling: pan-CK antibody is a cytoskeleton-specific antibody for tumor cells, possessing the advantages of high abundance and strong specificity, enabling precise localization of tumor cells. Poly HRP40 is a signal-enhancing enzyme label composed of five identical HRP homopolymers, capable of simultaneously covalently coupling with multiple streptavidin molecules, significantly amplifying the signal and improving labeling efficiency. Using HRP-directly labeled primary antibodies effectively avoids false positives from secondary antibodies, offers high specificity, eliminates the need for secondary antibodies, shortens experimental time, and is rapid and convenient.

[0048] 4) Tyrosamide Signal Amplification Technology: Utilizing tyrosamide signal amplification (TSA) technology, HRP catalyzes the production of a large number of enzymatic products from biotinylated tyrosine amides. These products bind to surrounding amino acid residues, thereby depositing a large amount of biotin in the target protein and its neighboring regions. This technology not only labels the target protein itself but also its neighboring proteins, thus maximizing the acquisition of the protein profile of tumor cells.

[0049] 5) Highly efficient enrichment strategy: Utilizing the high affinity of biotin-streptavidin, tumor cell proteins can be directly enriched from the protein system. The procedure is simplified, the reagents are safe, and the specificity is high. Attached Figure Description

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0051] Figure 1 This is a technical roadmap for the present invention.

[0052] Figure 2 This is a schematic diagram illustrating the technical principle of the present invention.

[0053] Figure 3 This is a high-confidence comparison of protein levels between Example 1 and Comparative Example 1 of the present invention.

[0054] Figure 4 The results show the comparison of protein subcellular localization between Example 1 and Comparative Example 1 of the present invention.

[0055] Figure 5 This is a comparison of the efficiency of enriching cellular components between Example 1 and Comparative Example 1 of the present invention.

[0056] Figure 6 The results show the enrichment pathway comparison between Example 1 and Comparative Example 1 of this invention. Detailed Implementation

[0057] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0058] The technical route of the present invention is as follows: Figure 1 The technical principle diagram is as follows: Figure 2 As shown.

[0059] Example 1: Detection of tumor cells

[0060] 1. Obtain FFPE slides of breast cancer for biotin-labeled tumor cells.

[0061] 1. Sample preparation

[0062] 1.1 Sample Selection: Select FFPE slides of breast cancer tissue, with a total area of ​​not less than 70 mm². 2 The thickness is 4μm.

[0063] 1.2 Pretreatment of sections: Place the selected FFPE sections into a baking machine and bake at 60°C for 1 hour to enhance the adhesion of the sections.

[0064] 2. Automatic staining machine dewaxing and sealing

[0065] 2.1 Instrument Preparation: Start the fully automatic intelligent staining machine and ensure that the following reagents have been added to the reagent tank: xylene, anhydrous ethanol, gradient alcohols (100%, 95%, 85%, 75%, 50%), pure water, EDTA antigen retrieval buffer (abcam, ab93680, pH 8.0), PBS buffer, endogenous peroxidase blocking solution (Yeasen, 36322ES60), and 5% BSA blocking solution.

[0066] 2.2 Dewaxing: After baking, place the sections into the slide rack of the staining machine. Set the dewaxing program: Xylene immersion: 10 minutes, repeated twice; Anhydrous ethanol immersion: 10 minutes, repeated twice; Gradient alcohol immersion: 100%, 95%, 85%, 75%, 50%, 5 minutes each. Run the program: Start the staining machine to automatically complete the dewaxing and gradient alcohol treatment.

[0067] 2.3 Hydration: Set the hydration program: Pure water immersion: 3 times, 1 minute each time. Run the program: Start the staining machine and automatically complete the hydration steps.

[0068] 2.4 Antigen retrieval: Transfer the slides to the antigen retrieval tank of the staining machine. Add EDTA antigen retrieval buffer (pH 8.0), ensuring the liquid completely covers the slides. Start the microwave heating program and perform antigen retrieval under the following conditions: heat at 100°C for 20 minutes, then turn off the microwave and allow it to cool to room temperature for 30 minutes.

[0069] 2.5 PBS washing: Set the PBS washing program: PBS immersion: 3 times, 5 minutes each time.

[0070] 2.6 Sample Region Marking: Remove the slide from the stainer and aspirate any surface liquid. Use an immunohistochemistry pen to circle the sample region on the slide. Place the slide back into the stainer's slide holder.

[0071] 2.7 Hydrogen peroxide blocking and sealing: Hydrogen peroxide blocking procedure: Add endogenous peroxidase blocking solution to cover the sample area and incubate at room temperature for 10 minutes. PBS washing procedure: Wash with PBS 3 times, 5 minutes each time. Sealing procedure: Add 5% BSA blocking solution to cover the sample area. Seal at room temperature for 1 hour.

[0072] 3. Direct labeling of primary antibody and antibody incubation

[0073] 3.1 Poly-HRP-labeled primary antibody: Protein A / G-Poly-HRP40 and pan-CK and EpCAM primary antibodies were mixed separately at a mass ratio of 2:1. After gentle mixing, the mixture was incubated at 4°C for 1 hour on a rotary mixer to promote binding.

[0074] 3.2 Antibody Incubation: The primary antibody directly labeled with Protein A / G-Poly-HRP40 was diluted to a working concentration of 1:100 with 1% BSA in PBS buffer. Protein A / G-Poly-HRP40-pan-CK and Protein A / G-Poly-HRP40-EpCAM were used for slide staining. The automated staining machine was programmed as follows: add the diluted primary antibody, completely covering the sample area, and incubate at 4°C for 16 hours. After incubation, the PBS washing program was set as follows: PBS wash: 3 times, 5 minutes each time.

[0075] 4. Biotin labeling: Add biotinylated tyrosine amide solution (15 μg / mL) to the reagent tank of the staining machine. Set the reaction program as follows: add the reaction solution dropwise to completely cover the sample area, and incubate at room temperature for 25 minutes. After incubation, set the PBS washing program as follows: PBS wash: 3 times, 5 minutes each time.

[0076] II. Extraction and Enrichment of Tumor Cell Proteins

[0077] 1. Freeze-drying: Place the washed sample into a freeze-drying centrifuge until the sample is completely dry.

[0078] 2. Protein Lysis: Using a clean scalpel, scrape the tissue section into a 1.5 mL EP tube. Add 100 μL of protein lysis buffer to the EP tube, mix thoroughly, and then sonicate on ice. The protein lysis buffer consists of: 0.1 M Tris-HCl, 0.1 M DTT, 2 mM EDTA, 1 mM PMSF, and pH 8.0. The sonication conditions are as follows: non-contact sonication at 540 W for 10 seconds, followed by a 10-second pause, for a total of 30 cycles. After sonicating for 5 minutes, remove the tube and centrifuge for 3-5 seconds. Repeat the sonication once more.

[0079] 3. Heating: Add 200 μL of 4% SDS solution to the lysed sample, vortex to mix, and incubate in a 95°C metal bath at 500 rpm for 90 min. After natural cooling, centrifuge at 13000 rpm for 15 min and collect the supernatant.

[0080] 4. SDS Washing: Add 1200 μL of pre-chilled acetone to the protein solution (acetone volume is 4 times that of the supernatant, final concentration is 80%), vortex to mix, centrifuge at 13000 rpm for 15 minutes at 4°C, repeat twice; after removing the supernatant, centrifuge at 1500 rpm at 4°C in a vacuum concentrator to resuspend the acetone. Resuspend in PBS. Quantify the protein using the BCA method.

[0081] 5. Purify biotinylated protein

[0082] 5.1 Prepare the buffer solution according to Table 2:

[0083] Table 2

[0084] Buffer Components Binding / Wash Buffer (pH 7.4) PBS elution buffer (pH 2.3) 0.2M Glycine + PBS

[0085] 5.2 Preparation of streptavidin magnetic beads:

[0086] a. Take the magnetic beads and remove the supernatant: Gently pipette to fully resuspend the streptavidin magnetic beads, take 50 μL and place it in a 1.5 mL centrifuge tube, and magnetically separate to remove the supernatant.

[0087] b. Washing the magnetic beads: Add 1 mL of wash buffer and gently resuspend the streptavidin magnetic beads by pipetting. Place on a magnetic rack for separation for at least 1 min, remove the supernatant by magnetic separation, and repeat once.

[0088] c. Resuspend the magnetic beads: Add 50 μL of PBS to resuspend the magnetic beads.

[0089] 5.3 Target protein binding to magnetic beads:

[0090] a. Incubate the prepared magnetic beads and the extracted protein separately on a rotary mixer at room temperature for 30 min;

[0091] b. Magnetic separation: After incubation, place the sample on a magnetic rack for 2-3 minutes to separate and remove the supernatant;

[0092] c. Washing: Add 500 μL of wash buffer containing 0.1% BSA, gently pipette to resuspend the magnetic beads, place on a magnetic rack for separation for 1 min, remove the supernatant, and repeat the washing 4-5 times.

[0093] 5.4 Elution: Add 50 μL of elution buffer, gently pipette to mix, and incubate at room temperature for 5 minutes on a rotary mixer. Then, separate on a magnetic rack for 1 minute, transfer the supernatant to a new centrifuge tube, and neutralize the elution buffer with an appropriate volume of Tris pH 8.0.

[0094] III. Proteolysis and Peptide Extraction

[0095] 1. Take protein samples and add them to 10kDa ultrafiltration tubes. Centrifuge at 14000g until there is no liquid on the membrane. Add 100μL of Urea buffer to the ultrafiltration tube and centrifuge at 14000g until there is no liquid on the membrane. Repeat twice.

[0096] 2. Reduction: Add 100 μL of 20 mM dithiothreitol and incubate at 56 °C with shaking for 1 hour.

[0097] 3. Alkylation: Add 1M iodoacetamide to a final concentration of 55mM and incubate at room temperature in the dark for 30 minutes.

[0098] 4. Centrifuge at 14000g until no liquid remains on the membrane. Add 100μL of Urea buffer to the ultrafiltration tube and centrifuge at 14000g until no liquid remains on the membrane. Repeat twice. Transfer the ultrafiltration membrane to a new collection tube.

[0099] 5. Enzymatic hydrolysis: Add 40 μL of 100 mM TEAB, add trypsin at a ratio of 1:100 to protein, and hydrolyze in a water bath at 37°C for 16 hours.

[0100] 6. Centrifuge at 14000g until no liquid remains on the membrane, add 50μL TEAB, centrifuge at 14000g until no liquid remains on the membrane, and collect the enzymatically digested peptides in the collection tube.

[0101] 7. Peptide Desalting: The enzymatically digested peptides are desalted using Strata-XL. The specific steps are as follows: activate the column with 1 mL of methanol, add 1 mL of 5% methanol for equilibration; dilute the sample 10:1 with 5% methanol and pass it through the column; rinse with 5% methanol to remove salt; finally, elute the peptides with 100% acetonitrile into a new 1.5 mL EP tube.

[0102] 8. Suspension: Seal the sample solution with aluminum foil and vacuum dry at 4℃ and 1500rpm.

[0103] IV. Mass Spectrometry Identification

[0104] 1. The two suspensions of peptide fragments were reconstituted with 20 μL of 0.1% FA and vortexed for 5 min to dissolve completely;

[0105] 2. Centrifuge at 15000 rpm and 4℃ for 30 min, collect the supernatant and add it to the built-in tube, then place it into the automatic sampler;

[0106] 3. Mass spectrometry detection.

[0107] Example 2: Detection of fibroblasts

[0108] The method steps in this embodiment are the same as in embodiment 1, except that:

[0109] Protein A / G-Poly-HRP40 and the primary antibodies aSMA and FAP were mixed at a mass ratio of 2:1 to obtain Protein A / G-Poly-HRP40-aSMA and Protein A / G-Poly-HRP40-FAP, and then proceeded with the subsequent antibody incubation steps.

[0110] Comparative Example 1: Traditional Method

[0111] The steps of this comparative example are the same as those in Example 1, except that:

[0112] Step 1 only involves dewaxing and rehydration, without heat-induced antigen retrieval, without HRP-labeled specific primary antibody labeling, and tyrosine signal amplification reaction; Step 2 differs from Example 1 in that the lysis buffer formulation is 20mM TrisHCl, 2% SDS, 200mM DTT, 20% glycerol, and 1% protease, and the incubation conditions are 100°C for 20 min, followed by incubation at 85°C for 2 h. The enriched protein is obtained by centrifugation after sedimentation at -20°C for more than 24 hours using acetone.

[0113] Example of effect

[0114] The mass spectrometry results of Example 1 and Comparative Example 1 were analyzed, and the high-confidence protein count results are as follows: Figure 3As shown in the figure. Compared with Comparative Example 1, Example 1 of the present invention shows significant advantages in protein analysis of FFPE samples. It enriches a greater number of peptides, especially when the FDR value is less than 0.01, the number of high-confidence proteins obtained is significantly increased compared with the traditional method, reflecting that the enrichment efficiency of the method of the present invention is higher and can provide richer and more effective information for subsequent research.

[0115] Subcellular localization analysis results are as follows Figure 4 As shown, the subcellular localization distribution of the proteins enriched in Example 1 is similar to that in Comparative Example 1, mainly concentrated in the cell nucleus, cytoplasm, cell membrane, and extracellular components. This result confirms that Example 1 has good feasibility, and its protein subcellular localization characteristics are consistent with the traditional scheme, indicating that the protein enrichment process in Example 1 did not significantly affect the subcellular localization properties of the proteins, thereby ensuring the integrity and accuracy of the protein information.

[0116] Component protein analysis results as follows Figure 5 As shown, Example 1 of this invention enriched a greater number of cellular component proteins than traditional methods, indicating that the protein enrichment process in Example 1 had a lower degree of contamination. By effectively reducing the loss of useful information due to contamination factors, the method in Example 1 can further improve the quality and reliability of protein analysis, providing purer and richer protein samples for protein research on FFPE samples, and facilitating more in-depth and accurate subsequent protein-related studies.

[0117] The results of enriched signaling pathways are as follows Figure 6 As shown, compared with Comparative Example 1, Comparative Example 2 exhibits higher representativeness and research value in enriching signaling pathways. This method successfully identified response pathways consistent with those found in previous mass spectrometry and proteomics analyses of microdissected FFPE lung cancer samples (NISHIMURA T, FUJII K, NAKAMURA H, et al. Protein co-expression network-based profiles revealed from laser-microdissected cancerous cells of lung squamous-cell carcinomas[J]. Scientific Reports, 2021, 11(1): 20209.), including "L13a-mediated translational silencing of CerμLoplasmin expression," etc. This further validates the effectiveness and scientific validity of the method of this invention, demonstrating that it can more comprehensively and accurately reveal key signaling pathways in tumors in proteomics research.

[0118] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A mass spectrometry method for identifying cell-specific proteins based on FFPE sample tissue sections, comprising the following steps: 1) After dewaxing and rehydration of FFPE sample tissue sections, heat treatment was performed to repair antigens; 2) Incubate the samples with a cell-specific antibody labeled with horseradish peroxidase; 3) Biotin labeling; 4) Prepare protein lysis samples; 5) Purification of streptomycin avidin; 6) Prepare mass spectrometry samples and perform mass spectrometry identification; The cells include tumor cells and fibroblasts.

2. The method according to claim 1, characterized in that: The area of ​​the slice mentioned in step 1) is 70-200 mm. 2 The thickness is 3-5 μm.

3. The method according to claim 2, characterized in that: The heat treatment described in step 1) includes: covering the slide with antigen retrieval buffer and then heating it; The temperature of the heat treatment is 80–120°C; The heating treatment time is 10 to 30 minutes.

4. The method according to claim 1, characterized in that: The cell-specific antibodies mentioned in step 2) include tumor cell-specific antibodies and fibroblast-specific antibodies; The tumor cell-specific antibodies include pan-CK and EpCAM; The fibroblast-specific antibodies include aSMA and FAP.

5. The method according to claim 1, characterized in that: The biotin-labeled sample in step 3) is treated with biotinylated tyrosine amide; The treatment concentration of the biotinylated tyrosine amide is 10–20 μg / mL; The treatment time for the biotinylated tyrosine amide is 20–30 min.

6. The method according to claim 5, characterized in that: Before step 3), endogenous peroxidase blocking and occlusion operations are performed.

7. The method according to claim 1, characterized in that: The method for preparing protein lysate samples in step 4) includes: protein treatment with lysis buffer and ultrasonic treatment; The lysis buffer includes Tris-HCl buffer, reducing agent, metal chelating agent, and protease inhibitor.

8. The method according to claim 1, characterized in that: The method for purifying streptavidin described in step 5) includes: enriching and purifying biotin-labeled proteins using streptavidin magnetic beads.

9. The method according to claim 1, characterized in that: Step 6) describes the preparation of the mass spectrometry sample, which includes the following steps: Reduction, alkylation, enzymatic hydrolysis, desalting, and drying.

10. The application of the mass spectrometry identification method according to any one of claims 1 to 9 in protein spectrometry detection.

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