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Cell non-targeted metabonomic analysis sample pretreatment method

A cell metabolism and analysis method technology, applied in analytical materials, scientific instruments, material separation, etc., can solve problems such as low extraction efficiency and incomplete metabolite extraction, and achieve high reproducibility, optimized extraction methods, and simple operation methods. Effect

Inactive Publication Date: 2019-05-24
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention mainly focuses on the extraction efficiency of metabolites in the sample pretreatment of cells based on GC-MS metabolomics analysis, and solves the technical problem of low extraction efficiency and incomplete extraction of metabolites in the prior art

Method used

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  • Cell non-targeted metabonomic analysis sample pretreatment method
  • Cell non-targeted metabonomic analysis sample pretreatment method
  • Cell non-targeted metabonomic analysis sample pretreatment method

Examples

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Effect test

Embodiment 1

[0051] 1) Cell number: Hep G2 cells grow adherently. Use DMEM medium containing 10% inactivated fetal bovine serum at 37°C, 5% CO 2 Cultured under conditions, digested with 0.25% trypsin, passaged once every 3 days at a ratio of 1:3, and the cells in the logarithmic growth phase were used for the experiment. Count the cells on a hemocytometer, and the number of experimental cells should reach at least 1×10 7 .

[0052] 2) Cell quenching method: put the cells on ice, absorb the extracellular culture medium, wash the cell culture dish with 1mL 4°C pre-cooled PBS 2-3 times, add -20°C pre-cooled quenching solution (3:2 methanol:water) 1 mL to quench the cells.

[0053] 3) Cell collection method: scrape the cells with a cell scraper, inhale into a 1.5mL Eppendorf centrifuge tube, centrifuge at 1000g for 5min, discard the supernatant, and collect the cells.

[0054] 4) Extraction of intracellular metabolites: add the cell sample obtained in step (3) to 1 mL of the prepared -20°C ...

Embodiment 2

[0058] The sample pretreatment for cell metabolome analysis was carried out according to the steps in Example 1, except that the method of cell collection in step (3) was replaced by 0.25% trypsin digestion for 2 minutes, and the cells were gently blown down with 4°C pre-cooled PBS.

Embodiment 3

[0060] Carry out pretreatment before the sample of cell metabolome analysis according to the steps of Example 1, the difference is that the derivatization reagent used in the second step of metabolite derivatization in step (5) is replaced by N-N- Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) solution.

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Abstract

The invention discloses a cell non-targeted metabonomic analysis sample pretreatment method and belongs to the field of chemical analysis and detection. The cell non-targeted metabonomic analysis sample pretreatment method comprises the following steps of (1) quenching cells with quenching liquid, and then collecting the cells, wherein the quenching liquid is a mixed solution of methanol and water; (2) adding extracting liquid into the cells obtained in the step (1) for extraction, wherein the extracting liquid is a mixed solution of acetonitrile, isopropanol and sterile water. When applied totreating samples, the cell non-targeted metabonomic analysis sample pretreatment method can ensure rich metabolite peak data and increase the mean number of metabolites up to 190 and the abundance tohigher than 60*108; meanwhile, the cell non-targeted metabonomic analysis sample pretreatment method can achieve high repeatability and accordingly provide model research for metabonomic research andaction mechanism elucidation on toxicology and efficacy with cells as the object of research.

Description

technical field [0001] The invention relates to a sample pretreatment method for cell non-targeted metabolomics analysis, belonging to the field of chemical analysis and detection. Background technique [0002] Cell metabolomics (Metabonomics / mebabolomics) is of great significance in biological research. Metabolomics is a science that studies the types, quantities and changes of metabolites (molecular weight <1000) of biological systems before and after being subjected to different external stimuli or disturbances (genetic modification or external environmental changes, etc.), and can analyze the local A metabolic pathway, which is an important part of a new type of systems biology. Compared with other omics, metabolomics can explore life activities at the level of metabolites. By analyzing the regulation of endogenous metabolites and the relationship with the overall biological status, biomarkers can be found, so as to find out the external environment. Possible pathwa...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/72G01N30/88
Inventor 孙秀兰崔方超纪剑孙嘉笛皮付伟张银志
Owner JIANGNAN UNIV
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