Muluneh Fashe   Muluneh Fashe Ph.D. is a postdoctoral research associate in Prof. Craig Lee's laboratory at the Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill. His research focuses on quantifying absolute concentrations of drug-metabolizing enzymes and transport proteins in membrane fractions of sandwich-cultured primary human hepatocytes in the absence or presence of inducers.

Using Skyline to Quantify Drug Metabolizing Enzyme and Transport Protein Concentrations in Sandwich-Cultured Primary Human Hepatocytes

The expression of drug-metabolizing enzymes (DMEs) and transporters in hepatocytes can be altered by various stimuli. Due to the large number of proteins belonging to the super-families of DMEs and transporters, traditional immunological-based methods of protein quantitation are inefficient, expensive, and laborious for performing a comprehensive quantitative proteomics analysis. We used a targeted quantitative proteomic isotope dilution nanoLC-MS/MS method to quantify absolute concentrations of over 70 phase I and II DME and transport proteins in membrane fractions isolated from sandwich-cultured primary human hepatocytes in the absence or presence of inducers. Using six representative DME and transport proteins (CYP3A4, POR, UGT1A1, BSEP, MRP2, and OATP1B1), we quantified absolute protein concentrations using the Skyline software and then using MultiQuant (SCIEX) software. Concentrations were compared between the platforms using correlation and Bland-Altman plot analyses. Results demonstrated the utility of the Skyline software for targeted quantitative proteomics data analysis in our experimental model. This presentation highlights the applicability of Skyline for processing and evaluating quantitative targeted absolute proteomic data for multiple DMEs and transporter proteins in the membrane fractions of human primary hepatocytes.