Method for pretreating research sample in blood non-target metabonomics

A metabolomics, non-target technology, applied in the field of sample pretreatment for simultaneous extraction of polar and non-polar metabolites in blood, can solve the problem of not being able to extract polar and non-polar metabolites of blood at the same time, and cannot be comprehensive and systematic Observing the specific changes and differences of blood metabolism changes, unable to meet the needs of metabolomics analysis of metabolites, etc., to achieve the effect of reducing the interference of high mass spectrometry matrix effects, reducing experimental operation steps, and reducing experimental errors

Active Publication Date: 2014-01-22
HARBIN MEDICAL UNIVERSITY
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Problems solved by technology

However, the shortcoming of these methods is to extract some metabolites in the blood. The organic solvent extraction method is mainly non-polar substances (lipids), and the solid phase extraction method removes the high concentration of lysophospholipids in the blood, while the Polar substances are rarely extracted
Therefore, the current sample pretreatment methods cannot simultaneously extract polar and non-polar metabolites in blood, and cannot meet the needs of metabolomics analysis of all metabolites in blood, and cannot comprehensively and systematically observe the specific changes and changes in blood metabolic changes. difference

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  • Method for pretreating research sample in blood non-target metabonomics

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Embodiment 1

[0022] Example 1 Pretreatment of Blood Non-target Metabolomics Research Samples

[0023] Follow the steps below:

[0024] (1) Take out the blood stored at -80°C, thaw at 4°C and vortex for 2 minutes, take 250 microliters of mixed plasma into a 2 ml centrifuge tube, add 750 microliters of methanol, vortex for 2 minutes, and centrifuge at 12,000 rpm at 4°C 10 minutes;

[0025] (2) Take the supernatant solution obtained after centrifugation in step (1) into another 2ml centrifuge tube, blow dry with nitrogen, and leave the solid residue for reconstitution;

[0026] (3) Mash the lower protein precipitate obtained after centrifugation in step (1), add 350 microliters of acetonitrile and water mixed solution, the volume ratio of acetonitrile and water in the acetonitrile and water mixed solution is 1:1, vortex for 2 minutes Finally, centrifuge at 12,000rpm at 4°C for 10 minutes to remove the protein in the plasma, and extract the polar substances and non-polar substances that have...

Embodiment 2

[0029] The mass spectrometry detection of sample after the pretreatment of embodiment 2

[0030] 1. Reagents

[0031] Acetonitrile, methanol (chromatographically pure), formic acid (chromatographically pure), and ultrapure water were prepared by a pure water instrument.

[0032] 2. Chromatographic separation conditions:

[0033] The liquid chromatography is the Acquity ultra-high performance liquid chromatography system of Waters Company of the United States. The chromatographic column is (BEH)C18 column (1.8μm, 2.1×100mm). Liquid chromatography conditions: mobile phase A is double-distilled water (0.1% formic acid solution), mobile phase B is acetonitrile, and the flow rate is 0.35mL / min. Elution with a linear gradient, the initial gradient is 2% acetonitrile and maintained for 0.5min , 0.5-1.5min acetonitrile 2%-20%, 1.5-6.0min acetonitrile 20%-70%, 6.0-10.0min acetonitrile 70%-98%, 10.0-12.0min acetonitrile 98% for 2min, and then the acetonitrile content decreased within...

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Abstract

The invention discloses a method for pretreating a research sample in blood non-target metabonomics. Polar and non-polar substances in serum can be extracted at the same time by adopting the method. Compared with an existing common sample pretreatment method, the method has the advantages that the number of extracted metabolites is remarkably increased, protein precipitate is dissolved by acetonitrile and water mixed solution, residues dried by nitrogen is re-dissolved, so that the dissolubility of non-polar substance lysophosphatide can be reduced on the one hand, and the high mass spectrum matrix effect interference caused by high-concentration lysophosphatide can be reduced; on the other hand, the experiment operation steps are reduced, the experiment errors are reduced, and a sample treated by adopting the method can be detected by a UPLC-Q-TOF MSMS (ultra-high-performance liquid chromatography / quadrupole time-of-flight mass spectrometry) as an analysis platform to obtain more metabolites in blood. Therefore, changes of blood metabolism can be favorably observed comprehensively, more reliable differential metabolites or biomarkers can be discovered, metabolic changes and change mechanism of a body can be further observed and researched, and the development of blood non-target metabonomics can be promoted.

Description

technical field [0001] The present invention relates to a new sample pretreatment method for blood non-target metabolomics research, in particular to a new method that uses UPLC-Q-TOF MSMS as an analysis platform and can simultaneously extract polar and non-polar components in blood. Methods for sample preparation of metabolites. Background technique [0002] Non-target blood metabolomics is a comprehensive investigation of biological systems stimulated or disturbed, using a series of technical means and detection methods to characterize the dynamic changes in the concentration and types of metabolites in the blood, and to observe changes in the body's metabolic pathways and body phases. Corresponding physiological and pathological changes. In order to clearly understand the changes in the overall metabolism of the human body, all the metabolites in the body fluid should be detected as much as possible, which requires a method to extract all the metabolites in the blood as ...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/06
Inventor 王茂清孙长颢李颖
Owner HARBIN MEDICAL UNIVERSITY
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