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Extraction and analysis method for free amino acid and fatty acid in adherent cells

A technology of free amino acids and free fatty acids, applied in the field of bioanalytical chemistry, to achieve the effects of small sample size, high sensitivity, and improved extraction efficiency

Active Publication Date: 2016-08-10
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Extraction and analysis method for free amino acid and fatty acid in adherent cells
  • Extraction and analysis method for free amino acid and fatty acid in adherent cells

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[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0025] (1) HepG2 was used as the model cell, seeded in a six-well plate, and the cell seeding density was 1.2×10 5 1 / mL, the amount of culture medium in each hole of the six-well plate is 2.5mL, the culture medium is DMEM (high glucose) liquid medium, containing 4500mg / l glucose, containing 10% fetal bovine serum and 1% volume fraction penicillin-streptomycin. After 24 hours of cultivation in the incubator (37°C, 5% CO 2 ), discard the culture medium, add 1mL water to each well to quickly wash and discard, and then add liquid nitrogen to quench. The amount of liquid nitrogen used is to fill each well of a six-well plate. After the liquid nitrogen has evaporated, add 0.5mL ultrapure water to each well and ultrasonically break for 5min, remove the six-well plate, place each well in a centrifuge tube, and freeze-dry at -20°C; after free...

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Abstract

The invention discloses an extraction and analysis method for free amino acid and fatty acid in adherent cells. The method mainly comprises the steps of: subjecting adherent cells to adherent culture in a culture tray, then subjecting a sample to rapid washing and conducting liquid nitrogen quenching, adding a small amount of water to perform ultrasonication, carrying out lyophilization, then adding MTBE for extraction, then adding water to carry out centrifugation and taking the supernatant, performing blow-drying with nitrogen, then using acetonitrile to make the volume to a constant level, and then carrying out liquid chromatography mass spectrometry (LC-MS) analysis on the fatty acid component, extracting the remaining part and further adding MTBE and methanol to conduct extraction, performing centrifugation, and taking the lower layer amino acid component to perform LC-MS analysis directly. The method provided by the invention adopts LC-MS to analyze free amino acid and fatty acid components, and utilizes a small sample size to realize simultaneous extraction of amino acid and fatty acid components. Compared with the conventional fatty acid GC-MS analysis method, the method has no need of derivatization, greatly simplifies the sample pretreatment process, and at the same time provides a reference method for extraction of adherent intracellular metabolites. The analysis method can be applied to analysis of free amino acid and fatty acid in other media.

Description

technical field [0001] The invention belongs to the field of bioanalytical chemistry, and relates to a method for extracting metabolites in adherent cells and an analysis method for amino acids and fatty acids based on liquid chromatography-mass spectrometry technology Background technique [0002] Intracellular amino acid and fatty acid components can participate in cellular energy metabolism pathways, and are the basic units for maintaining cellular carbon metabolism and nitrogen metabolism. Cells are stimulated by the external environment, and the composition of amino acids and fatty acids in the body will undergo corresponding changes after exposure to pollutants and drugs. In the field of biology, some amino acids have become markers of tumorigenesis, and metabolic disorders involving free fatty acids can also cause Diseases in the body. Therefore, the analysis of intracellular amino acid and fatty acid components is of great significance [0003] The analysis of amin...

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Application Information

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IPC IPC(8): G01N30/06
Inventor 耿柠波陈吉平张保琴张海军王菲迪
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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