Lipoprotein Quantitation and Function Core

脂蛋白定量和功能核心

• 批准号: 10711260
• 负责人: Tomas Vaisar
• 金额: $ 33.03万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10711260
• 关键词: Abbreviations; Apolipoprotein A-II; Apolipoproteins; Area; Binding; Bioinformatics; Biological; Biological Assay; Calibration; Cell Line; Chemicals; Cholesterol; Complex; Data; Data Set; Databases; Doctor of Philosophy; Electron Microscopy; Electrospray Ionization; Genes; Goals; Grant; Hamsters; Heterogeneity; High Density Lipoproteins; Human; Human Resources; In Vitro; Individual; Isotopes; Kidney; Knowledge; Laboratories; Lipoproteins; Liquid Chromatography; Mass Spectrum Analysis; Measurement; Methods; Mifepristone; Molecular; NMR Spectroscopy; Ontology; Particle Size; Pathway Analysis; Peptides; Phospholipids; Plasma; Process; Program Research Project Grants; Proteins; Proteome; Proteomics; Recombinant Proteins; Recombinants; Reproducibility; Research; Research Personnel; Services; Shotguns; Spectrometry; Standardization; Structure; System; Testing; Theoretical model; Universities; Washington; analytical method; analytical tool; cost effective; cost effectiveness; crosslink; data integration; gel electrophoresis; in vivo; instrumentation; interdisciplinary approach; ion mobility; kidney cell; mutant; particle; programs; protein protein interaction; reconstitution; stable isotope; stoichiometry; success; tandem mass spectrometry

ABSTRACT (Core C) High quality, consistent and reproducible quantitation and functional data are critical for the success of this Program Project. The overarching goal of the HDL Quantitation and Function Core (Core C) is to meet this need in a centralized and consistent manner across the entire Program. The Core is structured to provide the following services: 1) quantitation of HDL particle size and concentration (whether isolated from plasma or reconstituted HDL particles) for specific structural studies, using calibrated differential ion mobility analysis (cIMA); 2) mass spectrometric analyses to characterize the protein composition of HDL particles isolated or prepared by Program investigators, and chemical crosslinking strategies to support their structural studies; the Core will also use mass spectrometry to confirm the identity and purity of the recombinant proteins prepared by Core D; 3) a combination of accurate measurement of HDL particle concentration by calibrated IMA and accurate quantification of apolipoproteins by LC-MS/MS, including quantification of the stoichiometry of apolipoproteins on HDL particles; 4) bioinformatic support for interpreting HDL-P and proteomic data sets and for combining the data with information from Gene Ontology, protein-protein interactions, and pathway analysis; 5) measurements of cholesterol and phospholipid efflux, using baby hamster kidney cells conditionally expressing WT and mutant human ABCA1. Core C will be led by Tomas Vaisar, Ph.D., working closely with Chongren Tang, Ph.D., and it will be located at the University of Washington South Lake Union campus, where Project 1 is also located. Both labs have extensive, well-documented expertise in all areas of the Core’s proposed services. In the previous PPG cycle, this Core performed over 1000 analyses of HDL-P and size, over 500 LC-MS/MS analyses of HDL’s proteome, and crosslinking studies for characterizing and experimentally verifying theoretical models of HDL and ABCA1 structures. These services will be extended in this proposal. Collectively, they will enable cost-effective and rigorous testing of the hypotheses generated by the various Projects.

摘要（核心 C） 高质量、一致且可重复的定量和功能数据对于这项工作的成功至关重要 计划项目。 HDL 定量和功能核心（核心 C）的总体目标是满足这一目标 需要在整个计划中以集中且一致的方式进行。核心的结构是为了提供 以下服务： 1) HDL 颗粒大小和浓度的定量（无论是从血浆中分离还是从血浆中分离） 重组 HDL 颗粒）用于特定结构研究，使用校准的微分离子淌度分析 （cIMA）； 2) 质谱分析来表征分离的 HDL 颗粒的蛋白质组成或 由计划研究人员准备，以及支持其结构研究的化学交联策略；这 Core 还将使用质谱法来确认通过以下方法制备的重组蛋白的身份和纯度： 核心D； 3) 通过校准 IMA 精确测量 HDL 颗粒浓度和 通过 LC-MS/MS 准确定量载脂蛋白，包括化学计量的定量 HDL 颗粒上的载脂蛋白； 4) 解释 HDL-P 和蛋白质组数据集的生物信息学支持以及 用于将数据与来自基因本体、蛋白质-蛋白质相互作用和通路的信息相结合 分析; 5) 使用幼仓鼠肾细胞测量胆固醇和磷脂流出 条件表达 WT 和突变型人 ABCA1。核心 C 将由 Tomas Vaisar 博士领导，负责 与唐崇仁博士密切合作，该中心将位于华盛顿大学南湖联合分校 校园，项目 1 也位于其中。两个实验室在各个领域都拥有广泛且有据可查的专业知识 核心提议的服务。在之前的 PPG 周期中，该核心对 HDL-P 进行了 1000 多次分析 和大小，对 HDL 蛋白质组进行了 500 多次 LC-MS/MS 分析，以及用于表征和分析的交联研究 实验验证HDL和ABCA1结构的理论模型。这些服务将扩展至 这个建议。总的来说，它们将使对所产生的假设进行具有成本效益和严格的测试成为可能。 各种项目。