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Method for identifying intracellular small molecule metabolites

A technology of small molecule metabolites and identification methods, which is applied in the analysis of materials, material analysis by electromagnetic means, and measurement devices, etc., can solve the problems of difficult qualitative and other problems, and achieve easy operation, wide linear range, good accuracy and stability Effect

Inactive Publication Date: 2019-06-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

At present, intracellular metabolites are difficult to identify and a large number of standard samples are needed as controls, but the present invention provides a method for qualitative and quantitative analysis of intracellular small molecule metabolites without the need for standard products

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  • Method for identifying intracellular small molecule metabolites
  • Method for identifying intracellular small molecule metabolites
  • Method for identifying intracellular small molecule metabolites

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Embodiment Construction

[0025] The present invention is further described by the following specific examples, but does not limit the present invention.

[0026] 1) Metabolite extraction:

[0027] Take the 10cm culture dish of human liver cancer cells HepG2 in the logarithmic phase, wash it quickly with PBS, add liquid nitrogen to quench, and collect the cells with a cell scraper; add 1ml of 80% methanol / water extract placed in a 4°C refrigerator, and ultrasonicate in ice water for 8min , Vortex for 3 min, and repeat three ultrasonic vortex cycles; centrifuge at 8°C, 13000rmp / min for 5min, filter the supernatant with an organic phase filter, and transfer to vial for analysis.

[0028] 2) Retrieve the database:

[0029] Search for compounds with biological roles in the KEGG database, and obtain their Q-TOF MS / MS chromatogram information (collision energy, product ion and precursor ion mass, mass spectrometry detection mode) in the METLIN database, list the mass spectrometry detection mode, Table of i...

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Abstract

The invention provides a method for identifying intracellular small molecule metabolites. The qualitative analysis of the metabolites is performed according to an exact mass number and chromatogram matching, and the metabolite is relatively quantified according to an abundance of the chromatogram. The procedure of the analysis method is as follows: washing cultured adherent cells with PBS quicklyand then quenched with liquid nitrogen; then adding an extract liquor to extract all metabolites in the cells; searching KEGG and METLIN databases to obtain parameters of the metabolic pathway main metabolites such as the ion pair and collision energy and the like, and establishing a detection and analysis method of the small molecule metabolites; and performing qualitative and quantitative analysis on the intracellular metabolites by using tandem quadrupole linear ion trap high performance mass spectrometry (Q-Trap) combined with quadrupole-time of flight mass spectrometry (Q-TOF). The methodfor identifying the intracellular small molecule metabolites can be used for qualitative and quantitative analysis of metabolites in cell samples; the range of the identified metabolites detected bythe method is wider; and the linear range of the metabolites is wider; therefore, the problem that the qualitative analysis of the metabolites in the prior art is difficult and the analysis requires alarge number of standards for comparison is effectively solved.

Description

technical field [0001] The present invention is a method for identifying intracellular small molecule metabolites, specifically by searching the KEGG database (http: / / www.kegg.jp / kegg / ) and the METLIN database (https: / / metlin.scripps.edu / ) to obtain the structural information of the metabolites, the lipid-water partition coefficient and the MS / MS chromatogram information (accurate mass, abundance, collision energy) of the liquid chromatography-quadrupole time-of-flight mass spectrometer (LC-Q-TOF) and mass spectrometry detection mode), metabolites were qualitatively identified based on accurate mass and chromatogram matching, and intracellular metabolites were quantitatively analyzed using a quadrupole linear ion trap high-performance mass spectrometer (Q-trap). This method is different from the method of qualitative metabolites through standard products, and is a new way to identify small molecular metabolites in cells. Background technique [0002] Small molecular weight...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62
Inventor 罗云张保琴耿柠波王菲迪任晓倩张海军陈吉平
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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