title: Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay.
abstract: Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, P.R. China; Research Center for Drug Metabolism, School of Life Sciences, Jilin University, Changchun, P.R. China.
authors.forename: Xidong
authors.initials: X
authors.lastname: Liu
authors.forename: Lianghai
authors.initials: L
authors.lastname: Hu
authors.forename: Guangbo
authors.initials: G
authors.lastname: Ge
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Jing
authors.initials: J
authors.lastname: Ning
authors.forename: Shixin
authors.initials: S
authors.lastname: Sun
authors.forename: Ling
authors.initials: L
authors.lastname: Yang
authors.forename: Klaus
authors.initials: K
authors.lastname: Pors
authors.forename: Jingkai
authors.initials: J
authors.lastname: Gu
mesh_terms: D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D020033:Protein Isoforms; D003577:Cytochrome P-450 Enzyme System
keywords: Biomedicine; Cytochrome P450; MRM; Protein quantification; Stable isotope labeling