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Enzymolysis method for performing enzymolysis on tissue sample by using mild reducing agent and mass spectrometric detection method thereof

A technology for tissue samples and biopsy tissues, applied in the preparation of test samples, biological testing, measuring devices, etc., can solve the problems of tissue protein extraction difficulties, achieve the effect of shortening steps, high yield, and reducing the possibility of contamination

Pending Publication Date: 2021-10-26
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Based on special tissue samples, especially paraffin-embedded tissue samples, it is difficult to extract tissue proteins cross-linked by disulfide bonds

Method used

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  • Enzymolysis method for performing enzymolysis on tissue sample by using mild reducing agent and mass spectrometric detection method thereof
  • Enzymolysis method for performing enzymolysis on tissue sample by using mild reducing agent and mass spectrometric detection method thereof
  • Enzymolysis method for performing enzymolysis on tissue sample by using mild reducing agent and mass spectrometric detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] According to the formulation of the lysate in Table 3, the solid component was added to the liquid component for mixing, heated in a metal bath at 99° C. for 30 min, and then cooled to room temperature to obtain lysate 1-8.

[0081] table 3

[0082]

[0083]

Embodiment 2

[0085] Tissue samples: Take 10 white slices (non-stained) of paraffin sections (non-stained) of human esophageal biopsy normal tissue with a length of 1 cm, a width of 10 μm, and a thickness of 10 μm, scrape off the tissue, and place it in a different test tube with a volume of about 0.5 μL.

[0086] Obtaining protein and peptide information:

[0087] Step 1: Lysis of Tissue Samples

[0088] Add 50 μL of lysate 1-8 (Lysis buffer) to each test tube.

[0089] Step 2: Enzymatic hydrolysis:

[0090] 3 μg of trypsin was added to each test tube, and the mixture was shaken (1000 rpm) in an incubator at 37° C. for 18 hours.

[0091] Step 3: Go to DOC

[0092] Add 100% formic acid (FA) to each test tube to make the final concentration 2%, shake at room temperature for 3 min, centrifuge at 12000 rpm for 5 min, and take the supernatant into a new EP tube.

[0093] Step 4: Extraction

[0094] Add 350 μL of extract to each test tube, shake at temperature for 3 minutes, centrifuge at 1...

Embodiment 3

[0119] Take 10 white slices (non-stained) of paraffin sections (non-stained) of human colorectal biopsy tissue (carcinoma in situ) with a length of 1.5 cm, a width of 20 μm, and a thickness of 10 μm, and scrape off the tissue, with a volume of about 1 μL.

[0120] According to the operation in Example 2, the proteins and peptides were obtained and detected (150min gradient was selected for mass spectrometry detection, and the C18 analysis column length was 30cm). The detection results are shown in Table 6.

[0121] Table 6

[0122]

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Abstract

The invention discloses an enzymolysis method for performing enzymolysis on a tissue sample by using a mild reducing agent and a mass spectrometric detection method of the enzymolysis method. The invention provides a lysis solution. The lysis solution comprises a decontaminant with the final concentration of 1-4% by mass; the detergent is sodium deoxycholate and / or ammonium bicarbonate; 3-(2-carboxyl) phosphine hydrochloride with a final concentration of 5-20 [mu]M; 5-chloro-anthranilic acid with the final concentration of 20 to 60 mM; tris (hydroxymethyl) aminomethane-hydrochloric acid with the final concentration of 50 to 200mM; phenylmethylsulfonyl fluoride with the final concentration of 0.1 to 2 mM; and water. After a tissue sample is cracked by the cracking liquid, tissue protein and peptide fragments can be obtained at high yield.

Description

technical field [0001] The invention relates to an enzymolysis method for enzymatically hydrolyzing tissue samples with a mild reducing agent and a mass spectrometry detection method thereof. Background technique [0002] Microdissection is the most commonly used method to obtain biopsy tissue or a small amount of special tissue samples. However, the scope of this method is limited, and it is mostly used for cutting frozen tissues, which is time-consuming, laborious and expensive. However, for clinical samples, especially samples after minimally invasive surgery (such as biopsy tissue), the sample size is small and the amount is small, which can be used for research and medical detection and evaluation. Nowadays, the health and safety awareness of human beings for early detection and early diagnosis of their own diseases has been significantly improved, and minimally invasive surgery has gradually become popular. Therefore, the segmentation (regional cutting) technology and ...

Claims

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Application Information

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IPC IPC(8): G01N1/28G01N1/36C12P21/06G01N33/68
CPCG01N1/28G01N1/36C12P21/06G01N33/6848
Inventor 丁琛李玲玲秦兆宇王云之
Owner FUDAN UNIV
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