title: SDS-PAGE-free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes.
abstract: Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS-PAGE-free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1-butyl-3-methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D-SCX-RPLC-ESI-MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.
authors.affiliation: Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Dingyin
authors.initials: D
authors.lastname: Tao
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhao
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Ling
authors.initials: L
authors.lastname: Yang
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D014453:Glucuronosyltransferase / Q000032:analysis*; D006801:Humans; D007093:Imidazoles; D008862:Microsomes, Liver / Q000201:enzymology*; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014529:Uridine / Q000378:metabolism
chemical_list: C419324:1-butyl-3-methylimidazolium tetrafluoroborate; D007093:Imidazoles; D012997:Solvents; D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase; D014529:Uridine