Label-free quantitative proteomic methods for developmental pharmacology

项目摘要 药物代谢酶（DME）和药物转运蛋白（DT）的绝对蛋白定量是必不可少的 用于表征发育表达模式和确定环境和 监管影响。绝对DME和DT量化也是机械分析的关键输入。 基于生理学的药代动力学（PBPK）模型，旨在预测药物暴露和药物反应 在不同的群体中（例如，儿童、老年人和族裔群体）。传统上，绝对蛋白质定量 依赖于基于抗体的免疫定量（即，蛋白质印迹法），使用纯化的蛋白质作为校准 标准然而，基于蛋白质印迹的免疫定量具有许多局限性（例如，低吞吐量， 交叉反应性、窄的动态范围和差的再现性）。为了克服这些局限性，我们 假设DME和DT精确和有效的绝对蛋白定量可以使用 一种无标记LC-MSE定量蛋白质组学方法。无标记LC-MSE方法的主要优点 包括：1）不需要合成特征肽标准品; 2）蛋白质组标度绝对值 在单次LC-MSE运行中定量;和3）比其他定量蛋白质组学方法更具成本效益。 本论文的多学科研究包括两个具体目标：1）开发无标记的LC-MSE 用于在蛋白质组上绝对定量人肝脏中DME和DT的定量蛋白质组学方法 和2）评价一组个体供体中DME和DT的基于无标记LC-MSE的定量 人肝微粒体（HLM）和质膜组分（PMF），与基于LC-MRM的 靶向定量、mRNA表达和标记底物活性。成功发展 所提出的方法有望扩大生物分析工具箱，使人们能够更好地了解 发育表达模式和环境/调节对临床重要的影响，以及 被低估的，DME和DT。最终，这些知识将为儿童合理使用药物提供信息， 所有年龄段

Project Summary 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.