Protein Analysis Core

蛋白质分析核心

• 批准号: 9359530
• 负责人: Frank A Witzmann
• 金额: $ 13.92万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9359530
• 关键词: Affect; Apatites; Area; Biological Assay; Biopsy; Biotin; Calcium; Calcium Oxalate; Calgranulin A; Cells; Crystallization; Deposition; Disease; Disease Progression; Distant; Drug Targeting; Duct (organ) structure; Enzyme-Linked Immunosorbent Assay; Excision; Forms Controls; Human; Immunofluorescence Immunologic; Individual; Inflammation; Inflammatory; Injury; Innate Immune System; Investigation; Kidney Calculi; Label; Lasers; Learning; Limb structure; Link; Magnetism; Mass Spectrum Analysis; Measures; Microdissection; Microscopy; Molecular Profiling; Nephrolithiasis; Oxides; Pain; Paper; Papillary; Pathogenesis; Pathway interactions; Patients; Play; Plug-in; Predisposition; Process; Protein Analysis; Proteins; Resolution; Role; Sampling; Site; Sodium-Restricted Diet; Specificity; Staining method; Stains; Testing; Thinness; Tissue Sample; Tissues; Toll-Like Receptor Pathway; UMOD gene; Urine; Western Blotting; base; bikunin; biomarker panel; candidate marker; high salt diet; hypercalciuria; improved; inflammatory marker; interstitial; kidney interstitial tissue; link protein; multiple reaction monitoring; osteopontin; particle; potassium citrate; programs; protein expression; tongue papilla

Summary Core D will conduct all protein-related analyses for Projects 1, 2, and 3. Human urine samples will be collected by Projects 1 and 2 from highly selected and well-characterized stone-former and normal patient pools. Human kidney interstitial tissue will be collected by biopsy (Project 2) and subsequent laser microdissection (LMD) (Project 3) from those same patient pools. We will quantify inflammation in the urine samples using high sensitivity multiplexed magnetic particle-based enzyme-linked immunosorbent assay (MP-ELISA) assays in 1) idiopathic hypercalciuria (IH) of both papillary types and in normal subjects with high salt diet; 2) conditions of high and low sodium diets with and without potassium citrate; 3) whether high papillary injury scores correlate with inflammation in either urine or LMD samples; 4) patients with and without non-obstructive stone pain; and 5) patients with non-obstructive stone pain before and after surgical removal of collecting duct plugs. Exploiting our ability to obtain highly homogeneous LMD samples, we will use MP-ELISA to measure key toll-like receptor pathway-related molecules in the interstitium and thin limb cells away from and near plaque. We will measure osteopontin, THP, CD59, bikunin, and calgranulin (A, B and C) expression in regions of plaque and crystal-free interstitium near plaque and compare them to non-stone forming controls. We will assess oxidative injury in areas near or distant from plaque compared to plugging by targeting oxidized proteins and quantifying them by LFQMS of biotin hydrazide-derivatized protein carbonyls. We will also exploit our unique ability to combine sensitive LFQMS with the specificity of LMD to investigate currently unknown pathways or cellular mechanisms of plugging or plaque that could serve as biomarker panels or drug targets. We will analyze regions of interstitium from papillary biopsies that do and do not contain apatite plaque comparing affected areas to non- affected sites within the same individuals in the papillae with plaque and/or plugging calcium stone disease and extend these investigations to plaque overgrowth sites to identify and quantify proteins unique to Randall's plaque to examine the protein content of the plaque-stone interface

概括 核心 D 将为项目 1、2 和 3 进行所有蛋白质相关分析。将收集人类尿液样本 由项目 1 和 2 从经过精心挑选和充分表征的结石形成者和正常患者池中选出。人类 将通过活检（项目 2）和随后的激光显微切割（LMD）收集肾间质组织 （项目 3）来自相同的患者库。我们将使用高通量定量尿液样本中的炎症 灵敏度多重磁性颗粒酶联免疫吸附测定 (MP-ELISA) 测定 1) 乳头状类型和高盐饮食正常受试者的特发性高钙尿症（IH）； 2）条件 含或不含柠檬酸钾的高钠和低钠饮食； 3）高乳头损伤评分是否相关 尿液或 LMD 样本中有炎症； 4) 有或无非阻塞性石痛的患者；和 5) 手术切除集合管栓子前后出现非阻塞性石痛的患者。剥削 我们有能力获得高度均质的 LMD 样品，我们将使用 MP-ELISA 来测量关键的 Toll 样受体 远离和靠近斑块的间质和细肢细胞中的通路相关分子。我们将测量 斑块和无晶体区域中骨桥蛋白、THP、CD59、bikunin 和钙颗粒蛋白（A、B 和 C）的表达 斑块附近的间质，并将它们与非结石形成对照进行比较。我们将评估氧化损伤 与通过靶向氧化蛋白质并通过量化它们来堵塞相比，靠近或远离斑块的区域 生物素酰肼衍生的蛋白质羰基的 LFQMS。我们还将利用我们独特的能力将 具有 LMD 特异性的灵敏 LFQMS，用于研究当前未知的途径或细胞机制 可以作为生物标志物组或药物靶标的堵塞物或斑块。我们将分析以下地区 乳头状活检中含有和不含有磷灰石斑块的间质，将受影响区域与非磷灰石斑块进行比较 患有斑块和/或堵塞钙结石病的同一个体中乳头的受影响部位和 将这些研究扩展到斑块过度生长部位，以识别和量化兰德尔氏病特有的蛋白质 斑块以检查斑块-结石界面的蛋白质含量