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

项目概要/摘要 近几十年来，儿童和青少年的超重和肥胖患病率急剧增加。 成人，达到流行病比例。为了获得对肥胖病理生理学的新见解 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检测方法 可以轻松地在临床实验室中实施，以精确监测许多激素和 与肥胖密切相关的炎症标志物。不幸的是，目前的临床检测往往缺乏 重现性，特别是由于缺乏测定标准化而在不同临床实验室之间。 靶向质谱分析是一项很有前景的技术，可为血液蛋白提供稳健的多重检测。 因此，该应用程序的总体目标是开发和验证可重复和可转移的质量 基于光谱分析的多重蛋白质检测，可对一组肥胖标志物进行精确定量。 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（例如胰岛素、瘦素和其他激素）、促炎标记物（例如 C 反应蛋白）和 抗炎标记物（例如脂联素）。促进稳健性和可转移性的全面验证 对于这些测定，将进行实验室间测定验证。具体来说，目标 1 将重点关注初始检测 开发最佳配置并展示内源性多重检测 血浆中的分析物。目标 2 将集中于分析优化和完整的分析表征 再现性、稳定性、选择性、线性和定量限等方面。目标 3 将展示 通过对临床血浆试验队列中的内源分析物进行实验室间定量来分析的效用 来自正常体重和肥胖受试者以及减肥前后的肥胖个体的样本 针对胰岛素和瘦素等选定分析物，以成熟的免疫测定为基准。至 促进在其他实验室轻松实施经过验证的测定，所有测定数据包括 色谱数据和详细的标准操作程序将通过公共数据共享 存储库和分析门户。该项目将共同建立强大且易于转移的多重检测方法 这将使精确监测肥胖的病理生理学成为可能。

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.