Absolute quantification method of rat CYP450 enzyme mass spectrum

An absolute quantification and mass spectrometry technology, applied in the field of proteomics, can solve problems such as affecting the accuracy of quantification, and achieve the effect of facilitating method promotion, simple preparation method, and simple and easy sample processing process and quantitative method.

Inactive Publication Date: 2013-06-12
JILIN UNIV
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Problems solved by technology

It involves the determination of human CYP450 enzyme subtypes by biological mass spectrometry absolute quantification method; in the pretreatment of protein samples, solid phase extraction and concentration reconstitution are not carried out, which affects the quantitative accuracy of detection

Method used

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  • Absolute quantification method of rat CYP450 enzyme mass spectrum
  • Absolute quantification method of rat CYP450 enzyme mass spectrum
  • Absolute quantification method of rat CYP450 enzyme mass spectrum

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0053] Example 1: Determination of the content of five CYP450 enzyme subtypes in rat liver microsomal samples

[0054] Part 1: Preparation of Rat Liver Microsomal Samples

[0055] A. Administration induction process in rats

[0056] (1) Select two male rats with a body weight of 200g±10g, free to eat and drink;

[0057] (2) Purchase commercially available phenobarbital injection with a concentration of 100mg / ml and dilute to 15mg / ml with normal saline before use;

[0058] (3) The administration process is as follows

[0059] ① Phenobarbital induction group: intraperitoneal injection of 1 mL of phenobarbital in normal saline solution (15 mg / mL) for 3 consecutive days, once a day in the morning,

[0060] ②Blank control group: intraperitoneal injection of 1 mL of normal saline solution for 3 consecutive days, once a day in the morning;

[0061] B. Preparation of rat liver microsomes,

[0062] The two rats were operated as follows: the rats were killed by decapitation, the bl...

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Abstract

The invention discloses an absolute quantification method of rat CYP450 enzyme subtype mass spectrum, and belongs to the field of proteomic technology. Based on an LC / MS / MS technology, the method can be used for realizing absolute quantification of 5 CPY450 enzyme subtypes CYP2D3, CYP270, CYP2A1, CYP2D26 and CYP2B2 in the systems such as rat liver microsomes and the like at the same time by use of a strategy of quantifying enzyme with a specific peptide fragment generated by pancreatin hydrolysis of the CYP450 enzyme. The method disclosed by the invention mainly comprises the following steps of: pretreatment of a protein sample, preparation of a standard curve, preparation of a quality control sample and measurement of the protein sample. The method disclosed by the invention has good linear relationship, high accuracy and good reproducibility, is sensitive, precise and reliable, and can be applied to development of a new drug, study on drug metabolism and evaluation on mutual effect of drugs.

Description

technical field [0001] The invention belongs to the technical field of proteomics, and relates to an absolute quantitative method for simultaneously measuring five CYP450 enzyme subtypes in rats by mass spectrometry. Background technique [0002] After entering the body, the drug can be excreted in the form of one or more active or inactive metabolites through metabolic process. Due to genetic polymorphism and epigenetic factors, there are obvious racial differences and individual differences in the enzymes acting on the drug metabolism process. Therefore, the qualitative and quantitative analysis of drug-metabolizing enzymes has important reference value and significance for drug development, evaluation of drug metabolism process and study of drug-drug interactions. At present, the methods for quantitative analysis of enzymes mainly include probe drug method, Western bLotting method, 2-D electrophoresis method and mRNA detection method, but these methods have certain short...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
Inventor 胡良海刘喜东孙严彤杨艳顾景凯
Owner JILIN UNIV
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