Label-free quantitative proteomic methods for developmental pharmacology

项目概要 药物代谢酶 (DME) 和药物转运蛋白 (DT) 的绝对蛋白质定量至关重要 用于表征发育表达模式并确定环境和 监管影响。绝对 DME 和 DT 量化也是机械分析的关键输入 基于生理的药代动力学 (PBPK) 模型旨在预测药物暴露和药物反应 不同人群（例如儿科、老年人和种族群体）。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），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有许多局限性（例如，通量低、 交叉反应性、动态范围窄和重现性差）。为了克服这些限制，我们 假设可以使用以下方法实现 DME 和 DT 的准确有效的绝对蛋白质定量 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) 不要求合成特征肽标准； 2) 蛋白质组规模的绝对 单次 LC-MSE 运行中的定量； 3）比其他定量蛋白质组学方法更具成本效益。 拟议的多学科研究包括两个具体目标：1）开发无标记 LC-MSE 定量蛋白质组学方法，用于在蛋白质组上绝对定量人肝脏中的 DME 和 DT 规模； 2) 评估一组个体供体中基于无标记 LC-MSE 的 DME 和 DT 定量 人肝微粒体 (HLM) 和质膜组分 (PMF)，与基于 LC-MRM 的比较 目标定量、mRNA 表达和标记底物活性。成功开发了 所提出的方法有望扩展生物分析工具箱，从而更好地理解 发育表达模式和环境/监管对临床的重要影响，以及 DME 和 DT 未被充分认识。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.