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A method for preparing peptides from paraffin-embedded esophageal cancer tissue

A paraffin-embedded, esophageal cancer technology is applied in the field of peptide segment preparation, paraffin-embedded esophagus cancer tissue to prepare peptide segments, to achieve the effects of reducing sample loss, reducing clogging and increasing the number of detections

Active Publication Date: 2021-05-25
安阳市肿瘤医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From a technical point of view, FFPE tissue can only obtain slices with a thickness of several microns due to formalin fixation, dehydration, and wax sealing. great challenge

Method used

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  • A method for preparing peptides from paraffin-embedded esophageal cancer tissue
  • A method for preparing peptides from paraffin-embedded esophageal cancer tissue
  • A method for preparing peptides from paraffin-embedded esophageal cancer tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Comparison of fresh tissue and FFPE tissue using the method of the present invention

[0066] First, construct tumor-bearing mice. Esophageal cancer TE-8 cells in good growth state were injected subcutaneously at 1×107 / ml, 100 μl, into the 5-6 intercostal spaces of the chest wall and the lateral abdominal wall of BALB / c nude immunodeficient mice, and the status and tumor formation of the mice were observed daily . When the inoculation site touches a nodule with a diameter of about 1 cm and grows rapidly, the tumor tissue is stripped, and the tumor at one site is used as fresh tissue, and the peptide is extracted after freezing and grinding in liquid nitrogen, and the other site is routinely embedded in paraffin and sectioned. According to the method of the present invention, FFPE peptides are extracted, and the expression of proteins in the two samples is confirmed by Coomassie blue staining, such as figure 1 shown. According to high-resolution mass spectr...

Embodiment 2

[0068] Example 2: Comparison of FFPE samples between cancerous tissue and paracancerous tissue

[0069] The paraffin-embedded tissues of 10 patients with moderately differentiated esophageal cancer were collected (preserved for at least 1 year), and the results of histochemical staining were referred to (representative pictures in figure 2 , image 3 ,), to distinguish the cancerous tissue part from the paracancerous part, strip and collect them into 2ml centrifuge tubes according to the area, and collect 10 slice samples in each tube. According to the FFPE peptide extraction established by the method of the present invention and the optimized phosphorylated peptide extraction scheme, the peptides are obtained, and final high-resolution mass spectrometry detection and preliminary data analysis are performed.

[0070] Through the method of the present invention, it is found that there is a certain difference in the identified proteins between the cancer tissue and the para-ca...

Embodiment 3

[0072] Embodiment 3: FFPE protein dissolution buffer test used in the present invention

[0073] According to Broeckx's previous report, the protein lysate (20mM Tris hydrochloride, 200mM dithiothreitol, 2% sodium lauryl sulfate, 20% glycerol) can increase the protein recovery rate by 15%. times, the protein solution used in the present invention is different from it, specifically 100mM tris hydrochloride, 100mM dithiothreitol, 0.5% polyethylene glycol 20000, 1% lauryl sodium sulfate. So far, there is no report on the comparison of the extraction efficiency of the two lysates. Therefore, in this part, 5 paraffin-embedded mouse tumor tissues in Example 1 are used as objects. 20 slices are cut out from each tissue, and placed in 2 tubes (each 1.5 ml vials to collect 10 slices). Two groups of experiments were designed and carried out, wherein Group A established the FFPE peptide extraction scheme and optimized phosphorylated peptide extraction scheme according to the method of ...

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Abstract

A method for preparing peptides in paraffin-embedded esophageal cancer tissue, comprising the following steps: S1: dewaxing; S2: hydration; S3: sequentially adding 1ml of 70% alcohol and incubating at room temperature for 10 minutes; S4: dissolving Sample; S5: Transfer the protein solution obtained from S4 to a Barocycler matching test tube, place it on the fixed position of the Barocycler, set the program at 95°C, a total of 60 cycles, of which 40,000 psi in each cycle for 50 seconds, followed by 5,000 psi, Time 10 seconds; S6: Store the pellet in a ‑80°C refrigerator; S7: Precipitate protein with frozen acetone solution; S8: Centrifuge at 16000g at 4°C for 10 minutes to remove acetone; S9: Quantify protein; S10: Peptide digestion; S11: Phosphorylation Enrichment of peptides. The invention improves working efficiency and reduces sample loss caused by blockage.

Description

technical field [0001] The invention relates to the preparation of peptides in protein research, in particular to a method for preparing peptides in paraffin-embedded esophageal cancer tissue, and belongs to the field of biotechnology. Background technique [0002] Clinical proteomics research is to realize the molecular typing of disease populations and discover potential drug intervention targets and diagnostic markers by discovering the differences in the expression abundance of proteins and their modification levels between different patients and healthy people. Biopsy or surgically resected tissue is the basic sample that can be obtained during clinical treatment, including fresh / frozen storage and formalin-fixed and paraffin-embedded (Formalin-fixed and paraffin-embedding, FFPE) tissue samples. Among them, FFPE tissue can be stored stably for decades at room temperature, and is the gold standard for histopathological analysis. A century of development since the techno...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/36G01N1/30G01N33/68
CPCG01N1/30G01N1/36G01N33/6848G01N2001/302
Inventor 纪媛媛李军扩申敬伟王能超王巍云侯建彬
Owner 安阳市肿瘤医院