title: Distinction between human cytochrome P450 (CYP) isoforms and identification of new phosphorylation sites by mass spectrometry.
abstract: In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based on sequence homology. In the present work, we demonstrate the performance of a mass spectrometry-based strategy to simultaneously detect and differentiate distinct human Cytochrome P450 (CYP) isoforms including the highly similar CYP3A4, CYP3A5, CYP3A7, as well as CYP2C8, CYP2C9, CYP2C18, CYP2C19, and CYP4F2, CYP4F3, CYP4F11, CYP4F12. Compared to commonly used immunodetection methods, mass spectrometry overcomes limitations such as low antibody specificity and offers high multiplexing possibilities. Furthermore, CYP phosphorylation, which may affect various biochemical and enzymatic properties of these enzymes, is still poorly analyzed, especially in human tissues. Using titanium dioxide resin combined with tandem mass spectrometry for phosphopeptide enrichment and sequencing, we discovered eight human P450 phosphorylation sites, seven of which were novel. The data from surgical human liver samples establish that the isoforms CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2D6, CYP3A4, CYP3A7, and CYP8B1 are phosphorylated in vivo. These results will aid in further investigation of the functional significance of protein phosphorylation for this important group of enzymes.
authors.affiliation: Functional Proteomics, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum, Universitaetsstr. 150, ZKF, D-44801 Bochum, Germany. gorden.redlich@rub.de
authors.forename: Gorden
authors.initials: G
authors.lastname: Redlich
authors.forename: Ulrich M
authors.initials: UM
authors.lastname: Zanger
authors.forename: Stephan
authors.initials: S
authors.lastname: Riedmaier
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bache
authors.forename: Anders B M
authors.initials: AB
authors.lastname: Giessing
authors.forename: Martin
authors.initials: M
authors.lastname: Eisenacher
authors.forename: Christian
authors.initials: C
authors.lastname: Stephan
authors.forename: Helmut E
authors.initials: HE
authors.lastname: Meyer
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
mesh_terms: D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism / Q000601:surgery; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods
chemical_list: D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System