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Method for identifying amino-containing metabolite isomers through direct mass spectrometry and application of amino-containing metabolite isomers

A technology for isomers and metabolites, applied in the field of amino metabolite isomer identification, can solve the problems of complex processing, cumbersome operation, inability to distinguish isomers, etc., and achieves high accuracy, simple operation, good repeatability

Pending Publication Date: 2020-11-06
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chromatographic pretreatment is complicated and the operation is cumbersome, while spectroscopic method is difficult to identify isomers in complex samples
Conventional single-stage mass spectrometry cannot distinguish isomers, while fragmentation of two-stage mass spectrometry cannot identify isomers with stable structures and the same fragment ions
Ion mobility mass spectrometry can distinguish isomers through the unique ion mobility function according to the difference in the spatial structure of the compound, but the ion mobility mass spectrometry instrument is expensive and has not been widely used yet [4,5]

Method used

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  • Method for identifying amino-containing metabolite isomers through direct mass spectrometry and application of amino-containing metabolite isomers
  • Method for identifying amino-containing metabolite isomers through direct mass spectrometry and application of amino-containing metabolite isomers
  • Method for identifying amino-containing metabolite isomers through direct mass spectrometry and application of amino-containing metabolite isomers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The steps of the detection method for the identification of amino acid isomers are as follows:

[0043] (1) Derivatization reaction: prepare 100 μL of 50 mM sodium bicarbonate solution of 10 mM leucine, isoleucine, alanine, β-alanine, aspartic acid and iminodiacetic acid standards respectively, pH About 8, add 40μL 50mM DIPP-L-Ala-NHS acetonitrile solution under ice bath conditions, vortex mix each solution, react in ice bath for more than 10min, and then react for more than 10min at room temperature; vacuum the obtained mixture Remove acetonitrile with a centrifugal evaporation concentrator; add 10% formic acid solution to the residue to adjust the pH to 2-3; then use a reversed-phase Vac C18 desalting, collect the eluate and remove acetonitrile with a vacuum centrifugal evaporation concentrator, freeze-dry the obtained solid to obtain the amino metabolite derivatized product, add 5% acetonitrile to the obtained amino metabolite derivatized product to resuspend, Fina...

Embodiment 2

[0052] Example 2, the identification steps of amino-containing metabolite isomers in cells are as follows:

[0053] Step (1) Extraction of cell metabolites: take the cultured HepG2 cells out of the incubator, and aspirate the medium; wash the cells with 2 mL of 37°C PBS to remove the residual medium, then aspirate the PBS, and repeat this step twice; Then scrape the cells into a 15mL centrifuge tube, wash the culture plate with 1mL-20°C methanol, and put the washed methanol into the 15mL centrifuge tube; put the centrifuge tube into liquid nitrogen and freeze for 30s. Take out the centrifuge tube from liquid nitrogen, thaw on ice for 2 min, vortex for 15 s, sonicate for 30 s, incubate on ice for 10 min; vortex for 15 s, centrifuge at 3000 g for 5 min, and collect the supernatant. Add 500 μL methanol to the precipitate, repeat the above extraction steps, and collect the supernatant; then add 500 μL ultrapure water to the precipitate, and collect the supernatant according to the...

Embodiment 3

[0058] Example 3, the identification steps of amino-containing metabolite isomers in animal tissues are as follows:

[0059] Step (1) Extraction of tissue metabolites: Methanol / chloroform / water was formulated as an extractant in a ratio of 2:2:3. Weigh 10 mg of mouse liver tissue sample, and homogenize it in a mixed solution of 4 mL / g methanol and 2 mL / g water at 4°C. The homogenate was transferred into a small glass bottle, and 4 mL / g chloroform and 4 mL / g water were added and vortexed for 60 s. After standing on ice for 15 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C, take the supernatant and transfer it to a glass bottle, remove the methanol and water from the obtained liquid, and store the tissue metabolite powder in a -80°C refrigerator.

[0060] Step (2) Derivatization reaction of tissue metabolites: Add 100 μL of 50 mM NaHCO to 10 mg of tissue-extracted metabolite powder 3 After the solution was fully dissolved, it was centrifuged at 15 000 g for 15 min, an...

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Abstract

The invention discloses a method for identifying amino metabolite-containing isomeride by direct mass spectrometry, which comprises the following steps: amino metabolite derivatization reaction: preparing an amino metabolite standard solution from amino metabolite, and adding a derivatization reagent solution to perform derivatization reaction to obtain an amino metabolite derivatization product;amino metabolite derivatization product detection: adding a solvent into the prepared amino metabolite derivatization product to prepare a heavy suspension, diluting the obtained heavy suspension, andcarrying out mass spectrometry sample introduction detection; mass spectrometry conditions are as follows: a positive ion mode is adopted, and an ion source is ESI; MS and MS / MS scanning is automatically carried out according to a data mode during operation of the mass spectrometer, and 10-50 precursor ions with the highest abundance are obtained through HCD fragmentation separation for analysisand identification. According to the method, the N-diisopropyl phosphate-L-alanine-N-hydroxysuccinimide ester is applied to identification of the isomeride for the first time, and the method for identifying the amino metabolite isomeride has high accuracy and wide applicability.

Description

technical field [0001] The invention belongs to the field of identifying isomers of amino metabolites, and in particular relates to a method for identifying isomers of amino metabolites by direct mass spectrometry and an application thereof. Background technique [0002] Amino-containing metabolites refer to small-molecule metabolites containing amino groups, including amino acids, polypeptides, neurotransmitters, and polyamines, which are involved in various metabolic pathways in life activities. In the study of metabolomics, the qualitative and quantitative analysis of amino-containing metabolites in complex biological samples is of great significance [1,2] . Because amino-containing metabolites include functional groups such as amino, methyl, and carboxyl, these substances have the phenomenon of isomers, which increases the difficulty of detection in complex biological samples. [0003] Currently, methods for the detection of isomers include chromatography, spectroscopy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/72
CPCG01N30/72
Inventor 高祥刘星星董继扬
Owner XIAMEN UNIV
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