title: A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes.
abstract: The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV <or=27%). The MS-based method strongly correlated with the immunoquantification method (r(2)>or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.
authors.affiliation: School of Pharmacy, The University of North Carolina at Chapel Hill, NC 27599, USA. michael.wang@unc.edu
authors.forename: Michael Zhuo
authors.initials: MZ
authors.lastname: Wang
authors.forename: Judy Qiju
authors.initials: JQ
authors.lastname: Wu
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Dennison
authors.forename: Arlene S
authors.initials: AS
authors.lastname: Bridges
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Hall
authors.forename: Sally
authors.initials: S
authors.lastname: Kornbluth
authors.forename: Richard R
authors.initials: RR
authors.lastname: Tidwell
authors.forename: Philip C
authors.initials: PC
authors.lastname: Smith
authors.forename: Robert D
authors.initials: RD
authors.lastname: Voyksner
authors.forename: Mary F
authors.initials: MF
authors.lastname: Paine
authors.forename: James Edwin
authors.initials: JE
authors.lastname: Hall
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000737:chemistry*; D008875:Middle Aged; D040901:Proteomics / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D035501:Uncertainty
chemical_list: D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A