Quantitative shot-gun proteomics and MS-based activity assay for revealing gender differences in enzyme contents for rat liver microsome

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Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/51519

Title:	Quantitative shot-gun proteomics and MS-based activity assay for revealing gender differences in enzyme contents for rat liver microsome
Authors:	Huang,HJ;Tsai,ML;Chen,YW;Chen,SH
Contributors:	通訊工程學系
Keywords:	GROWTH-HORMONE REGULATION;SEX-DEPENDENT EXPRESSION;CYTOCHROMES P450;MASS-SPECTROMETRY;MEMBRANE-PROTEINS;GENE-EXPRESSION;MOUSE-LIVER;FEMALE RATS;DRUG;IDENTIFICATION
Date:	2011
Issue Date:	2012-03-27 18:55:11 (UTC+8)
Publisher:	國立中央大學
Abstract:	Liver microsomes are subcellular fractions that contain many metabolizing enzymes for drugs and endogeneous compounds. Some of these enzymes are regulated by sex hormonal control and exhibit sex-dependent expression pattern and metabolizing speed. Studying these enzymes, however, are complicated by the presence of isoforms such as cytochrome P450 (CYP450), which families share more than 50% amino acid identities. In this study, we applied quantitative shot-gun proteomics approach coupled with stable-isotope dimethyl labeling, two-dimensional reversed-phase peptide separation and tandem mass spectrometry (MS) to explore the gender-dependent expression of rat liver microsomal proteins. A total of 391 proteins were identified and quantified by this approach, and 56% of quantified proteins were enzymes. Although shot-gun approach is rarely used for identifying protein isoforms, we identified 53 isoforms by at least one unique peptide including 21 isoforms of CYP450s. Moreover, by quantitative and statistics assessment, we were able to classify them into 28 male dominant enzymes including CYP2C12 CYP2C11, CYP2C13, CYP2B3, CYP2C11, CYP2C70 and CYP3A2 which are known to be male specific, 21 female dominant enzymes including CYP2A1, CYP2C7, CYP2C12, CYP2D26, alcohol dehydrogenase 1, carboxylesterase 3, glutathione S-transferase, liver carboxylesterase 4, UDP-glucuronosyltransferase 281, and glyceraldehyde-3-phosphate dehydrogenase which are known to be female specific; and 125 sex-independent enzymes. However, most of the sex specificities revealed from this study, such as the male specificity of CYP2D1, were novel and not yet reported. We then conducted a mass spectrometry-multiple reaction mode (MS-MRM) based enzyme activity method to determine the catalyzing rate of CYP2D1 in male and female liver microsomes using carteolol as its specific substrate. The reaction rate catalyzed by CYP2D1 in female rats was determined to differ significantly with the rate in male rats. Moreover, the ratio (female/male) of reaction rate (0.68) was found to correlate with their relative protein abundance (0.72). This study revealed novel sex dependences of many rat liver enzymes and also demonstrated a unique MS-based analytical platform that could identify novel iso-enzymes and further quantify their abundance and enzyme activity. (C) 2011 Elsevier B.V. All rights reserved.
Relation:	JOURNAL OF PROTEOMICS
Appears in Collections:	[Department of Communication Engineering] journal & Dissertation

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