Absolute Quantification of Cytochrome P450 and Uridine-Diphosphate Glucuronosyl Transferase Isoforms by Proteomics-based Approach

A strategy for the absolute quantitation of metabolic enzymes in rat liver microsomes was developed based on shotgun-based proteomics approach. Rat liver microsomes were digested with trypsin, and drug metabolizing enzymes were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) using standard curves based on custom synthesized specific peptides for each enzyme. The assays for cytochrome P450 (CYP450) and uridine diphosphoglucuronosyl transferase (UGT) showed good linearity (r > 0.995) with lower limits of quantitation of 10 nM. A synthetic isotope labeled specific peptide was then used as internal standard to determine UGT1A1 by stable isotope dilution method. The labeled peptide behaved similarly to the unlabeled peptide in LC-MS/MS and the assay was linear in matrix solution. The UGT1A1 concentration was detected by the labeled peptide method to be 18.2 pmol mg−1 protein compared with 17.3 pmol mg−1 protein obtained by the standard curve method. Although the consistent results were achieved by the two methods, the stable isotope dilution method was more convenient and more suitable for high throughput determinations in complex systems.

Specific peptides from seven P450 and UGT isoforms were selected for the absolute protein quantification in rat liver microsomes by shotgun-based proteomic approach.Figure optionsDownload as PowerPoint slide