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Detection method and detection kit for quantifying rat CYP2E1 enzyme based on characteristic peptide fragment

A technology for quantitative detection and characteristic peptides, which is applied in the field of biological detection and protein, can solve the problems of high quantitative lower limit, insufficient sensitivity, and lack of method verification, etc., and achieve the effect of convenient detection

Inactive Publication Date: 2018-12-07
余鹏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method does not use actual samples for verification when screening peptides. It only uses software to screen out specific peptides. Whether it is unique and has a high enough response in the instrument cannot be confirmed in the early screening. At the same time, the current Some methods have not been validated after the method is developed, and the sample quantification is completed only by standard curve, the lower limit of quantification is high, and the sensitivity is not enough

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  • Detection method and detection kit for quantifying rat CYP2E1 enzyme based on characteristic peptide fragment
  • Detection method and detection kit for quantifying rat CYP2E1 enzyme based on characteristic peptide fragment
  • Detection method and detection kit for quantifying rat CYP2E1 enzyme based on characteristic peptide fragment

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Embodiment 1

[0061] This example provides a detection method for the quantitative detection of CYP2E1 enzymes using characteristic peptides, including the following steps: artificially synthesized according to the pre-designed quantitative peptides and labeled peptides; the quantitative peptides include the first quantitative peptide and the second quantitative peptide The amino acid sequence of the first quantitative peptide and the second quantitative peptide is shown in SEQ ID NO.1-2; the amino acid sequence of the first quantitative peptide SEQID NO.1: FINLVPSNLPHEATR; the second quantitative peptide SEQ Amino acid sequence of ID NO.2: GIIFNNGPTWK.

[0062] The labeled peptides include the first labeled peptide and the second labeled peptide. The first labeled peptide and the second labeled peptide are labeled with isotopes. The isotope labeling method of the first labeled peptide is: FINL( 13 C 6 15 N 1 )VPSNLPHEATR; the isotope labeling method of the second labeled peptide is GII(...

Embodiment 2

[0078] This example provides a detection method for the quantitative detection of CYP2E1 enzymes using characteristic peptides, including the following steps: artificially synthesized according to the pre-designed quantitative peptides and labeled peptides; the quantitative peptides include the first quantitative peptide and the second quantitative peptide segment, the amino acid sequence of the first quantitative peptide SEQ ID NO.1: FINLVPSNLPHEATR; the amino acid sequence of the second quantitative peptide SEQ ID NO.2: GIIFNNGPTWK.

[0079] The labeled peptides include the first labeled peptide and the second labeled peptide. Both the first labeled peptide and the second labeled peptide are labeled with an isotope. The isotope labeling method of the first labeled peptide is: FINL( 13 C 6 15 N 1 )VPSNLPHEATR; the isotope labeling method of the second labeled peptide is GII( 13 C 6 15 N 1 ) FNNGPTWK. And prepare the first quantitative peptide working solution, the second...

Embodiment 3

[0095] This embodiment provides a detection method for quantitatively detecting rat CYP2E1 enzymes based on characteristic peptides, which includes the following steps: artificially synthesizing pre-designed quantitative peptides and labeled peptides; quantitative peptides include the first quantitative peptide and the second quantitative peptide. Quantitative peptides, the amino acid sequence of the first quantitative peptide SEQ ID NO.1: FINLVPSNLPHEATR; the amino acid sequence of the second quantitative peptide SEQ ID NO.2: GIIFNNGPTWK. The labeled peptides include the first labeled peptide and the second labeled peptide. Both the first labeled peptide and the second labeled peptide are labeled with an isotope. The isotope labeling method of the first labeled peptide is: FINL( 13 C 6 15 N 1 )VPSNLPHEATR; the isotope labeling mode of the second labeled peptide is GII( 13 C 6 15 N 1 ) FNNGPTWK. And prepare the first quantitative peptide working solution, the second qua...

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Abstract

The invention provides a detection method and a detection kit for quantifying a rat CYP2E1 enzyme based on a characteristic peptide fragment and belongs to the technical field of biodetection and proteins. The method comprises the following steps: screening a characteristic peptide fragment of the rat CYP2E1 enzyme as a quantifying peptide fragment and artificially synthesizing the peptide fragment as an analytical reference substance, and synthesizing a stable isotope labeled peptide fragment as an interior label; carrying out linear regression by taking a specific value of the quantifying peptide fragment and the interior label peptide fragment as a vertical coordinate and the quantifying peptide of a series of standard blood serum samples as a transverse coordinate to obtain a regression equation; releasing the quantifying peptide fragment through enzymolysis of a to-be-detected sample; adding the labeled peptide fragment for feeding analysis; and obtaining the precise content of the rate CYP2E1 enzyme in the samples according to the linear regression equation. Two quantifying peptide fragments are selected to synthesize corresponding labeled peptide fragments as interior labels, thereby achieving a purpose of mutual verification. The detection kit can simply a sample pre-treatment step, so that the rat CYP2E1 enzyme is detected conveniently.

Description

technical field [0001] The invention relates to the field of biological detection and protein technology, in particular to a detection method and a detection kit for quantifying rat CYP2E1 enzyme based on characteristic peptide segments. Background technique [0002] CYP450 enzymes are the main enzymes in phase I metabolism of drugs, and their content is one of the important factors affecting the activity. Existing quantification of drug metabolizing enzymes generally uses immunological methods, such as Western blot. However, most CYP450 enzymes have homology, and it is difficult to find specific antibodies. Therefore, it is necessary to establish a more accurate and sensitive quantitative method. [0003] Different research institutions have different quantitative methods for metabolic enzymes, mainly including quantitative RT-PCR, semi-quantitative RT-PCR to quantitatively analyze mRNA at the transcription level, or immunohistochemical techniques (IHC) and Western Blot ...

Claims

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Application Information

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IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/045G01N2030/8818G01N2030/8822
Inventor 余鹏邱朝辉孟凡奇蒋蕾丁尧张可
Owner 余鹏
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