BioAnalytical Chemistry Core B: Innate reponses to microbial infection

生物分析化学核心 B：对微生物感染的先天反应

基本信息

批准号：
8305639
负责人：
Bradford Wayne Gibson
金额：
$ 49.78万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8305639
关键词：
Analytical Chemistry Area Bacteria Biochemistry Carbohydrate Chemistry Carbohydrates Cell membrane Chemistry Collaborations Complex Complex Mixtures Data Detergents Francisella tularensis Glycoconjugates Glycolipids Human Immune Immune response Infection Institutes Label Link Lipid Chemistry Lipids Lipoprotein (a)Lipoproteins Mass Spectrum Analysis Mediating Membrane Membrane Lipids Membrane Microdomains Methods Modification Molecular Molecular Conformation Monitor Natural Immunity O Antigens Organelles Peptides Phospholipids Phosphorylation Plasma Polysaccharides Post-Translational Protein Processing Preparation Process Program Research Project Grants Protein Analysis Protein Chemistry Proteins Proteome Proteomics Recruitment Activity Regulation Resistance Resources Stable Isotope Labeling Staging Structure Technology Vesicle Work base calmodulin-dependent protein kinase II capsule cell type comparative immunoregulation interest lipooligosaccharide microbial mutant neutrophil protein complex research study response

项目摘要

The BioAnalytical Chemistry Core B will provide expert bio-analytical chemistry support to the three projects of this Program Project Grant, 'Innate Responses to Microbial Infection'. These areas include mass spectrometry, protein chemistry & proteomics (Part I), and carbohydrate & lipid chemistries (Part II). In Part I, a comparative analysis of proteins, including discovery-based and quantitative mass spectrometry based proteomic methods, will be carried out on human neutrophil preparations, capsule-like material (CLM). and various traget proteins of interest. For Project 1 (Apicella). we will examine CLM perparations for the presence of a putative lipoprotein anchor and. if present, its identity and lipid modifications will be elucidated. As part of Project 2 (Nauseef). we will target human cell types that are critical to the reguation or dysregulation of the innate immune response and identify changes in protein composition and expression contained in detergent-resistant membrane microdomains or lipid rafts. For these experiments, various proteomic approaches will be used, including non-lableing and ITRAQ labeling strategies. Lastly, in Project 2 and 3 (Nauseef and Allen), a targeted examination of posttranslational modifications will be carried out with an emphasis on phosphorylation for several key host proteins invloved in immune regulation, i.e., p47phox, CaMKII and PKCa. For these experiment, a multireaction monitoring approach using stable isotope-labeled peptides will be employed for targeted quantitation. In Part II, we will use our expertise in mass spectrometry and carbohydrate and lipid chemistries for the structural characterization of host plasma phospholipids and capsule-like material (CLM). With Dr. Apicella (Project 1), we will carry out an exhaustive analysis of CLM preparations to identify its polysaccharide structure and its membrane anchor, likely a diacylglycerolphosphate-linked moiety. For Project 2 (Nauseef). an examination of the changes in phospholipid composition in recruited neutrophils will be undertaken. All experiments will be carried out in the Buck Institute's proteomics and mass spectrometry facility. This facility, of which Dr. Gibson is Director, is capable of both high-throughput and targeted analysis of complex mixtures of proteins, as well as detailed analysis of complex of carbohydrates and lipids. Dr. Gibson will also work with the NMR facility at lowa. who will acquire 1 and 2D NMR data from CLM preparations

生物分析化学核心 B 将为该计划项目拨款的三个项目“对微生物感染的固有反应”提供专家生物分析化学支持。这些领域包括大众光谱测定法、蛋白质化学和蛋白质组学（第一部分）以及碳水化合物和脂质化学（第二部分）。在第一部分中，将对人类中性粒细胞制剂、胶囊样材料（CLM）进行蛋白质比较分析，包括基于发现和基于定量质谱的蛋白质组学方法。和各种感兴趣的目标蛋白。对于项目 1 (Apicella)。我们将检查 CLM 制剂中是否存在假定的脂蛋白锚。如果存在，其身份和脂质修饰将被阐明。作为项目 2 (Nauseef) 的一部分。我们将针对对调节或调节至关重要的人类细胞类型先天免疫反应失调，并识别耐去垢剂膜微域或脂筏中所含蛋白质组成和表达的变化。对于这些实验，将使用各种蛋白质组学方法，包括非标记和 ITRAQ 标记策略。最后，在项目 2 和 3（Nauseef 和 Allen）中，将对翻译后修饰进行有针对性的检查，重点是参与免疫调节的几个关键宿主蛋白的磷酸化，即 p47phox、CaMKII 和 PKCa。对于这些实验，将采用使用稳定同位素标记肽的多反应监测方法进行靶向定量。在第二部分中，我们将利用我们在质谱、碳水化合物和脂质化学方面的专业知识来表征宿主血浆磷脂和胶囊状材料 (CLM) 的结构。我们将与 Apicella 博士（项目 1）一起对 CLM 制剂进行详尽的分析，以确定其多糖结构及其膜锚，可能是磷酸二酰基甘油连接的部分。对于项目 2（Nauseef）。将检查招募的中性粒细胞中磷脂成分的变化。所有实验都将在巴克研究所的蛋白质组学和质谱设施中进行。该设施由吉布森博士担任主任，能够对复杂的蛋白质混合物进行高通量和有针对性的分析，以及对碳水化合物和脂质复合物进行详细分析。吉布森博士还将在洛瓦的 NMR 设施中工作。谁将从 CLM 制剂中获取 1 维和 2D NMR 数据