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Method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry

An epoxide and LC/MS technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of poor specificity, difficult to measure large quantities of samples, etc., and achieve the effect of high sensitivity

Active Publication Date: 2020-05-22
JIANGSU PROVINCIAL HOSPITAL OF TCM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, both of these methods are semi-quantitative methods that depend on specific antibodies, which have poor specificity and are not easy to measure large batches of samples.

Method used

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  • Method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry
  • Method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry
  • Method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry

Examples

Experimental program
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Embodiment 1

[0036] Example 1: Determination of the concentration of sEH protein in rat heart, liver and kidney tissues

[0037] 1. Experimental materials and instruments

[0038] Materials: DFLLGAFQMK standard peptides and EKDFLLGAFQMK peptidomimetics: Jiangsu GenScript Biotechnology Co., Ltd., content greater than 98.5%; DFLLGAFQMK* isotope peptide stock solution: Thermo Scientific, USA, content greater than 95%; BCA kit, DDT, IAA and Pancreatin: Thermo Scientific; Acetonitrile and methanol: Merck Company, HPLC grade; Formic acid: ACS Company, HPLC grade; Water was prepared by Millipore Milli-Q Advantage A10 ultrapure water machine.

[0039] Instrument: API 4000 LC-MS / MS LC-MS, including: (1) Pump Agilent 1260G1312A; (2) Column Oven Agilent 1260G1316A; (3) Auto Sampler Agilent 1260G1367E; (4) Massspectrometer API4000; (5) Value Valco 2-Position, controlled by Analyst 1.6 Software; WH-2 Mini Vortex Mixer (Shanghai Huxi Analytical Instrument Factory); MS-100 Constant Temperature Mixer (Hangzhou ...

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Abstract

The invention discloses a method for detecting soluble epoxide hydrolase by liquid chromatography-mass spectrometry. The method comprises the following steps: (1) screening a specific polypeptide of the soluble epoxide hydrolase from tissues, and showing the amino acid sequence of the specific polypeptide as SEQ ID NO.1; taking specific polypeptide as a standard substance, carrying out detecting by utilizing a mass spectrum isotope internal standard quantitative method and a liquid chromatography-mass spectrometry method, and establishing a standard curve by taking the concentration of the standard substance as an abscissa and the ratio of a quantitative daughter ion peak area to an isotope internal standard peak area as an ordinate; and (2) determining the concentration of the specific polypeptide in a sample by the liquid chromatography-mass spectrometry method, and then carrying out converting and calculating to obtain the concentration of the soluble epoxide hydrolase in the sample. According to the present invention, a peptide fragment with characteristics of stable enzymolysis and high enzymolysis efficiency is selected from the representative characteristic peptide fragmentwith stable property as the quantitative peptide fragment, such that advantages of accuracy, precision, sensitivity and the like are realized, and the sEH in the rat tissue can be effectively, accurately and quantitatively analyzed.

Description

Technical field [0001] The invention belongs to a method for absolute protein quantification, and specifically relates to a method for detecting soluble epoxide hydrolase (sEH) by liquid mass spectrometry (LC-MS / MS). Background technique [0002] Epoxide hydrolase (EH) is a class of enzymes ubiquitous in mammals, insects, plants, and microorganisms. It can stereoselectively add water molecules to epoxides to generate corresponding 1, 2 -Diol. Soluble epoxide hydrolase (sEH) is a member of the epoxide hydrolase family. It was discovered by Gill et al. in 1973 when they were studying the metabolism of terpenoid epoxides similar to insect juvenile hormones. It is named mainly in the soluble components of cells such as cytoplasm and peroxisomes. In mammalian systems, sEH has an α / β hydrolase folding structure and can hydrolyze trans-substituted epoxides and a series of aliphatic epoxides converted from fatty acids. These epoxides are important signaling molecules in the body, whic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/54G01N30/60G01N30/72
CPCG01N30/02G01N30/06G01N30/34G01N30/54G01N30/6052G01N30/72G01N2030/045G01N2030/067
Inventor 许美娟陈影姜超袭晓韵吴婷吴坚张倩储继红居文政戴国梁
Owner JIANGSU PROVINCIAL HOSPITAL OF TCM
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