Multiplex Mass Spectrometric Protein Assays for Precise Monitoring of the Pathophysiology of Obesity

项目摘要/摘要 近几十年来，超重和肥胖的流行率在儿童和 成人，达到流行比例。为了对肥胖的病理生理学有新的认识 为了更好地个性化患者护理，迫切需要开发可靠的多重蛋白质分析，以 可以在临床实验室中轻松实施，以实现对许多激素和 炎症标志物与肥胖密切相关。不幸的是，目前的临床检测往往缺乏 由于缺乏检测标准，重复性差，特别是在不同的临床实验室之间。 靶向质谱学是一种很有前途的技术，可以提供强大的血液蛋白质多重分析。 因此，该应用程序的总体目标是开发和验证可重现和可转移的质量 基于光谱学的多重蛋白质分析，能够对一组肥胖标记物进行精确量化。 该小组将涵盖肥胖病理生理学的多个方面，包括葡萄糖稳态和能量。 平衡(如胰岛素、瘦素和其他激素)、促炎标志物(如C反应蛋白)，以及 抗炎标记物(如脂联素)。以促进对健壮性和可转移性的全面验证 在化验中，将进行实验室间化验验证。具体地说，目标1将专注于初始测试 最优构型的研究进展及对内源基因多重可信检测的论证 血浆中的分析物。目标2将集中在测试优化和全面的测试表征在 重现性、稳定性、选择性、线性和定量限度。目标3将演示 通过实验室间对临床血浆试点队列中的内源性分析物进行量化的分析方法的应用 正常体重和肥胖者，以及肥胖者减肥前后和 对照成熟的免疫分析方法对选定的分析物，如胰岛素和瘦素进行比较。至 促进在其他实验室轻松实施经过验证的化验，所有化验数据包括 色谱数据和详细的标准操作程序将通过公共数据共享 存储库和化验门户。总而言之，该项目将建立强大的、易于转移的多重分析 这将使对肥胖的病理生理进行精确监测成为可能。

PROJECT SUMMARY/ABSTRACT The prevalence of overweight and obesity has increased dramatically in recent decades in both children and adults, reaching an epidemic proportion. In order to gain new insights into the pathophysiology of obesity towards better personalized patient care, there is a critical need to develop reliable multiplex protein assays that can be easily implemented in clinical laboratories to enable precise monitoring of many hormones and inflammatory markers closely associated with obesity. Unfortunately, current clinical assays often lack of reproducibility, especially between different clinical laboratories due to the lack of assay standardization. Targeted mass spectrometry is a promising technology for providing robust multiplex assays for blood proteins. Therefore, the overall objective of this application is to develop and validate reproducible and transferable mass spectrometry-based multiplex protein assays for enabling precise quantification of a panel of obesity markers. The panel will cover multiple aspects of obesity pathophysiology, including glucose homeostasis and energy balance (e.g., insulin, leptin, and other hormones), pro-inflammatory markers (e.g., C-reactive protein), and anti-inflammatory markers (e.g., adiponectin). To facilitate full validation of the robustness and transferability of the assays, an inter-lab assay validation will be pursued. Specifically, Aim 1 will be focused on initial assay development for optimal configurations and on demonstrating confident multiplex detection of endogenous analytes in blood plasma. Aim 2 will be centered on assay optimization and full assay characterization in the aspects of reproducibility, stability, selectivity, linearity, and limit of quantification. Aim 3 will demonstrate the utility of the assays through inter-lab quantification of endogenous analytes in a pilot cohort of clinical plasma samples from normal weight and obese subjects, and obese individual before and after weight loss and benchmarking against well-established immunoassays for selected analytes such as insulin and leptin. To facilitate the easy- implementation of the validated assays in other laboratories, all assay data including chromatographic data and detailed standard operating procedures will be shared through public data repositories and assay portals. Together, the project will establish robust and easy-to-transfer multiplex assays that will enable precise monitoring of the pathophysiology of obesity.