GC-MS-based plant non-targeted metabolomics sample pretreatment method

A sample pretreatment and metabolomics technology, applied in the field of GC-MS-based plant non-targeted metabolomics sample pretreatment, can solve the problems of less mass spectrometry information, poor reproducibility, and non-general screening, etc., to achieve Improving research efficiency, strengthening data exchange, and simple and fast operation

Active Publication Date: 2016-08-17
上海鹿明生物科技有限公司
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Problems solved by technology

[0004] The purpose of plant metabolomics experiments is to conduct qualitative and quantitative analysis of as many metabolites as possible in plant samples at the same time, but because there are many types of metabolites in plant samples and their contents vary greatly, the dynamic range of metabolite concentrations is wide and complicated. The degree is relatively high, and the metabolites are easy to react when heated, resulting in structural changes
In addition, plant tissues contain a lot of fat-soluble secondary metabolites. These substances are various and have high boiling points, and it is difficult to reduce the boiling point by derivatization methods, so they are not suitable for detection by GC-MS (gas chromatography-mass spectrometry)
The mass spectrometry information in the GC-MS database (Fiehn database, NIST database, etc.) of these substances is scarce and difficult to characterize. It is not the main purpose of general screening, and it is easy to interfere with the analysis of primary metabolites.
In addition, the pathway analysis of secondary metabolites is complicated. There are few secondary metabolic pathways in the KEGG database, and there is a lack of comparison basis for secondary metabolic pathways. Therefore, as many primary metabolites in plant samples as possible are extracted in the pretreatment. , and exclude the interference of fat-soluble secondary metabolites
However, the existing GC-MS-based plant non-targeted metabolomics sample pretreatment method has poor reproducibility and poor versatility. The extraction process did not achieve low-temperature extraction as much as possible, and did not strictly control the Temperature, high temperature during operation can easily lead to changes in the structure of metabolites, and the extraction of metabolites is not sufficient, it is difficult to achieve high-throughput and full-range detection, and it is difficult to detect substances with large differences in properties or contents at the same time. The sex is not high, the fat-soluble secondary metabolites cannot be eliminated, and the interference cannot be avoided

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  • GC-MS-based plant non-targeted metabolomics sample pretreatment method

Examples

Experimental program
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Effect test

Embodiment 1

[0031] The pretreatment of embodiment 1 tea tree leaf (sugar content 1~5%)

[0032] The following steps were used to pretreat fresh tea tree leaves and perform GC-MS detection. The parallel experiments were repeated 6 times (1A-1F).

[0033] The specific steps of preprocessing are:

[0034] 1) Accurately weigh 60 mg of tea tree leaves, put them into a 1.5 mL centrifuge tube; add two small steel balls, 360 μL methanol at 4 °C and 40 μL internal standard methanol solution (L-2-chloro-phenylalanine, 0.3 mg / mL), placed in a -80°C refrigerator for 2 min; put into a grinder for grinding (60 Hz, 2 min), removed from the grinder, and ultrasonically extracted for 30 min;

[0035] 2) Add 200 μL of chloroform, vortex in a grinder (20 Hz, 2 min), add 400 μL of water, vortex in a grinder (20 Hz, 2 min), and ultrasonically extract for 30 min;

[0036] 3) Centrifuge at low temperature for 10 minutes (14000rpm, 4°C), take 200-700μL supernatant, put it into a glass derivatization bottle, and...

Embodiment 2

[0045] The pretreatment of embodiment two watermelon stems (sugar content about 2%)

[0046] The fresh watermelon stems were pretreated and detected by GC-MS using the following steps, and the parallel experiments were repeated 6 times (2A-2F).

[0047] The specific steps of preprocessing are:

[0048] 1) Accurately weigh 60mg of watermelon stem, put it into a 1.5mL centrifuge tube; add two small steel balls, 360μL-20℃ ethanol and 40μL internal standard ethanol solution (L-4-chlorophenylalanine, 0.3 mg / mL), placed in a refrigerator at -20°C for 2 min; put into a grinder for grinding (60 Hz, 2 min), take it out from the grinder, and ultrasonically extract for 30 min;

[0049] 2) Add 200 μL ether, put it into a grinder and vortex (20Hz, 2min), add 400μL water, put it into a grinder, vortex (20Hz, 2min), and ultrasonically extract for 30min;

[0050] 3) Centrifuge at low temperature for 10min (14000rpm, 16°C), take 200-700μL of the supernatant, put it into a glass derivative bo...

Embodiment 3

[0058] Example 3 Pretreatment of Zanthoxylum bungeanum root (sugar content is about 1%)

[0059] The fresh Zanthoxylum bungeanum root was pretreated and detected by GC-MS using the following steps, and the parallel experiments (3A-3F) were repeated 6 times.

[0060] The specific steps of preprocessing are:

[0061] 1) Accurately weigh 60mg of Zanthoxylum bungeanum root, put it into a 1.5mL centrifuge tube; add two small steel balls, 360μL-10℃ acetone and 40μL internal standard acetone solution (3,4-dichlorophenylalanine, 0.3mg / mL), placed in a refrigerator at -10°C for 2min; put into a grinder for grinding (60Hz, 2min), took it out of the grinder, and ultrasonically extracted for 30min;

[0062] 2) Add 200 μL of ethyl acetate, vortex in a grinder (20 Hz, 2 min), add 400 μL of water, vortex in a grinder (20 Hz, 2 min), and ultrasonically extract for 30 min;

[0063] 3) Centrifuge at low temperature for 10 minutes (14000rpm, 10°C), take 200-700μ of the supernatant, put it into...

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Abstract

The invention discloses a GC-MS-based plant non-targeted metabolomics sample pretreatment method. The method comprises the steps of 1) mixing up a plant sample, an internal standard substance and a hydrophilic organic solvent, wherein the hydrophilic organic solvent is pre-cooled to be -20 to 4 DEG C; cooling the obtained mixture to be -80 to -10 DEG C, grinding, crushing and conducting the supersonic extraction in the ice-water bath; 2) then respectively adding a lipophilic organic solvent and water, uniformly mixing up, conducting the supersonic extraction in the ice-water bath, conducting the high-speed centrifugation at the temperature of 0 to 16 DEG C, obtaining the aqueous phase and evaporating; 3) adding an oximation reagent, and conducting the oximation reaction at the temperature of 30 to 45 DEG C; 4) finally adding a derivatization reagent and n-hexane, and conducting the derivatization reaction at the temperature of 60 to 80 DEG C. According to the technical scheme of the invention, based on the above pretreatment method, the primary metabolites of a plant sample can be fully extracted, so that the abundant metabolite spectrum data information can be obtained. Meanwhile, both the change of metabolites during the extraction process, and the pollution of liposoluble substances to a gas chromatographic column can be avoided as much as possible. Therefore, the better sample reproducibility is realized.

Description

technical field [0001] The invention belongs to the field of plant sample pretreatment methods, and in particular relates to a GC-MS-based non-targeted plant metabolomics sample pretreatment method. Background technique [0002] At present, plant metabolomics has evolved from a concept full of hypotheses into an academic field with rapid development and great research value. Unlike bacteria and yeast, which contain only a few hundred metabolites, about 100,000 plant secondary metabolites are known. However, plant metabolomics research is still in its infancy, and has broad application prospects in the fields of species identification, transgenic plant identification, metabolic pathways and gene function research. [0003] Through the study of metabolome, we can not only understand the changes of plants under different environmental conditions, but also study the composition and content of metabolites in different parts or periods of the same plant. Metabolic profiling can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06
CPCG01N30/06G01N2030/062
Inventor 白娴舒烈波彭章晓杨卓郭峻杰
Owner 上海鹿明生物科技有限公司
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