title: Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics.
abstract: Cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGT) are major determinants in the pharmacokinetics of most drugs on the market. To investigate their impact on intestinal and hepatic drug metabolism, we developed and validated quantification methods for nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) that have been shown to be of clinical relevance in human drug metabolism. Protein quantification was performed by targeted proteomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based determination of enzyme specific peptides after tryptic digestion using in each case stable isotope labelled peptides as internal standard. The chromatography of the respective peptides was performed with gradient elution using a reversed phase (C18) column (Ascentis(®) Express Peptide ES-C18, 100mm×2.1mm, 2.7μm) and 0.1% formic acid (FA) as well as acetonitrile with 0.1% FA as mobile phases at a flow rate of 300μl/min. The MS/MS detection of all peptides was done simultaneously with a scheduled multiple reaction monitoring (MRM) method in the positive mode by monitoring in each case three mass transitions per proteospecific peptide and the internal standard. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity (0.25-50nM), within-day and between-day accuracy and precision, digestion efficiency as well as stability. Finally, the developed method was successfully applied to determine the CYP and UGT protein amount in human liver and intestinal microsomes. The method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to quantify clinically relevant human CYP and UGT enzymes.
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Gröer
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Busch
authors.affiliation: Department of General, Visceral, Thoracic and Vascular Surgery, University Medicine, Greifswald, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Patrzyk
authors.affiliation: Department of General, Visceral, Thoracic and Vascular Surgery, University Medicine, Greifswald, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Beyer
authors.affiliation: Department of General, Visceral, Thoracic and Vascular Surgery, University Medicine, Greifswald, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Busemann
authors.affiliation: Department of General, Visceral, Thoracic and Vascular Surgery, University Medicine, Greifswald, Germany.
authors.forename: C D
authors.initials: CD
authors.lastname: Heidecke
authors.affiliation: Department of Experimental and Clinical Pharmacology, Pomeranian Medical University, Szczecin, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Drozdzik
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Siegmund
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany. Electronic address: stefan.oswald@uni-greifswald.de.
authors.forename: S
authors.initials: S
authors.lastname: Oswald
mesh_terms: D000328:Adult; D000368:Aged; D002138:Calibration; D056148:Chromatography, Reverse-Phase / Q000592:standards; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D014453:Glucuronosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes; D007583:Jejunum / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008875:Middle Aged; D010449:Peptide Mapping; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000592:standards; D055815:Young Adult
chemical_list: D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase
keywords: CYP; LC–MS/MS; Protein quantification; UGT