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Absolute quantitative method for biomass spectra of CYP450 enzyme hypotypes

A biological mass spectrometry and absolute quantification technology, applied in the field of proteomics, can solve problems such as unreliability, improve method accuracy, eliminate chromatographic behavior and mass spectrometry response, and eliminate errors

Inactive Publication Date: 2013-06-05
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Third, protein expression is regulated by multiple mechanisms, and it is not very reliable to use the level of mRNA expression to represent its corresponding protein content

Method used

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  • Absolute quantitative method for biomass spectra of CYP450 enzyme hypotypes
  • Absolute quantitative method for biomass spectra of CYP450 enzyme hypotypes
  • Absolute quantitative method for biomass spectra of CYP450 enzyme hypotypes

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Determination of the content of four CYP450 enzyme subtypes in mixed human liver microparticles prepared by a certain company

[0045] The main steps are as follows:

[0046] A. Protein sample pretreatment

[0047] (1) Reduction of protein samples

[0048] ①Add 100μl protein sample to 1.5ml EP tube;

[0049] ② Add 5 μl denaturant and vortex to mix;

[0050] ③ Add 10 μl reducing agent and vortex to mix;

[0051] ④ Incubate at 60°C for 1 hour to obtain a reaction solution.

[0052] (2) Blocking of cysteine

[0053] ①Add 5 μl cysteine ​​blocking agent to the reaction solution obtained in step (1), and vortex to mix;

[0054] ② Incubate at room temperature for 10 minutes.

[0055] (3) Trypsin digestion of protein samples

[0056] ① Add 100 μl of digestion buffer to the reaction solution obtained in step (2);

[0057] ② Add 10 μl trypsin solution, vortex to mix;

[0058] ③Incubate overnight at 37°C (12 to 16h).

[0059] The solution formula involved in th...

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Abstract

The invention relates to an absolute quantitative method for biomass spectra of CYP450 enzyme hypotypes, and belongs to the technical field of proteomics. In the method, on the basis of liquid chromatography (LC) / mass spectrometry (MS) / MS, the absolute quantification of four CYP450 enzyme hypotypes, namely CYP1A2, CYP2B6, CYP3A4 and CYP3A5 in systems such as liver microsome and the like is realized simultaneously by a strategy that CYP450 generates specific peptide quantitative enzymes by pancreatic enzyme hydrolysis. The method mainly comprises the following steps of: preprocessing a proteinsample, preparing a standard curve, measuring the protein sample and preparing a quality control sample. The absolute quantitative method has excellent linear relations, is high in accuracy and repeatability, is sensitive, precise and reliable, and can be used for medicinal metabolism and the evaluation of the mutual effect of medicaments.

Description

technical field [0001] The invention belongs to the technical field of proteomics and relates to a biological mass spectrum absolute quantitative method for simultaneously measuring four human CYP450 enzyme subtypes. Background technique [0002] P450 enzymes are important drug-metabolizing enzymes in the body. While P450 enzymes metabolize drugs, drugs can also induce or inhibit P450 enzymes. Among them, four P450 enzymes, CYP1A2, CYP2B6, CYP3A4, and CYP3A5, participate in about 85% of drug metabolism in vivo. The qualitative and quantitative analysis of P450 enzymes has important reference value and significance for evaluating drug metabolism process and drug interaction. Existing P450 enzyme quantitative analysis methods mainly include probe drug method, Western Blot method, and mRNA detection method. However, the above method has poor specificity and low sensitivity, and can only give the relative amount of P450 enzymes, but not the absolute content of P450 enzymes, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/72G01N30/06
Inventor 顾景凯何群张芸辉杨艳孙严彤史美云曾苏蒋惠娣
Owner JILIN UNIV
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