Absolute protein quantification of drug metabolizing enzymes (DMEs) and drug transporters (DTs) is essential for the characterization of developmental expression patterns and determination of environmental and regulatory influences. Absolute DME and DT quantification also are critical inputs for mechanistic physiologically-based pharmacokinetic (PBPK) models aimed at predicting drug exposure and drug response in diverse populations (e.g., pediatric, geriatric and ethnic groups). Traditionally, absolute protein quantification has relied on antibody-based immunoquantification (i.e., Western blot) using purified proteins as calibration standards. However, Western blot-based immunoquantification has many limitations (e.g., low throughput, cross-reactivity, narrow dynamic range and poor reproducibility). To overcome these limitations, we hypothesize that accurate and efficient absolute protein quantification of DMEs and DTs can be achieved using a label-free LC-MSE quantitative proteomic method. The major advantages of a label-free LC-MSE method include: 1) no requirement for synthetic signature peptide standards; 2) proteome-scaled absolute quantification in a single LC-MSE run; and 3) more cost-effective than other quantitative proteomic methods. The proposed multidisciplinary research consists of two specific aims: 1) develop a label-free LC-MSE quantitative proteomic method for absolute quantification of DMEs and DTs in human liver on a proteome scale; and 2) Evaluate label-free LC-MSE?based quantification of DMEs and DTs in a panel of individual donor human liver microsomes (HLM) and plasma membrane fractions (PMF), comparing to LC-MRM-based targeted quantification, mRNA expression and marker substrate activities. Successful development of the proposed method is expected to expand the bioanalytical toolbox, enabling a better understanding of developmental expression patterns and environmental/regulatory influences on clinically important, as well as under-recognized, DMEs and DTs. Ultimately, this knowledge will inform the rational use of drugs in children of all ages.
Absolute protein quantification of drug metabolizing enzymes (DMEs) and drug transporters (DTs) is essential for the characterization of developmental expression patterns and determination of environmental and regulatory influences. Absolute DME and DT quantification also are critical inputs for mechanistic physiologically-based pharmacokinetic (PBPK) models aimed at predicting drug exposure and drug response in diverse populations (e.g., pediatric, geriatric and ethnic groups). Traditional Western blot-based immunoquantification method has many limitations, e.g., low throughput, cross-reactivity, narrow dynamic range and poor reproducibility. Therefore, development of a new protein quantification method for DMEs and DTs that overcomes these limitations is highly desirable. In this application, a label-free LC-MSE quantitative proteomic method is proposed to provide accurate and efficient absolute quantification of DMEs and DTs on a proteome scale. Successful development of the proposed method is expected to expand the bioanalytical toolbox, enabling better understanding of developmental patterns and environmental/regulatory influences on clinically important, as well as under-recognized, DMEs and DTs, and ultimately, the application of this knowledge to the rational use of drugs in children of all ages.
