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

• 批准号: 7920685
• 负责人: Bradford Wayne Gibson
• 金额: $ 50.43万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7920685
• 关键词: Analytical Chemistry; Area; Arts; 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

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将为该计划项目资助的三个项目“微生物对先天反应”提供专家生物分析化学支持。这些领域包括质量 光谱学，蛋白质化学和蛋白质组学（第一部分），碳水化合物和脂质化学（第二部分）。 在第一部分中，蛋白质的比较分析，包括基于发现和基于定量质谱的蛋白质组学方法，将在人中性粒细胞制剂，胶囊样材料（CLM）上进行。 和各种目标蛋白质。项目1（Apicella）。我们将检查CLM制备物中是否存在假定的脂蛋白锚。如果存在，将阐明其身份和脂质修饰。 作为项目2（恶心）的一部分。我们将针对对调节至关重要的人类细胞类型， 研究先天性免疫应答的失调，并鉴定抗洗涤剂膜微区或脂筏中所含的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（恶心和艾伦）中，将进行翻译后修饰的靶向检查，重点是参与免疫调节的几种关键宿主蛋白质的磷酸化，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，我们将利用我们在质谱和碳水化合物和脂质化学方面的专业知识，对宿主血浆磷脂和胶囊样物质（CLM）进行结构表征。与Apicella博士（项目1）一起，我们将对CLM制剂进行详尽的分析，以确定其多糖结构及其膜锚，可能是磷酸二酰甘油连接的部分。项目2（恶心）。 将对募集的中性粒细胞中磷脂组成的变化进行检查。 所有的实验都将在巴克研究所的蛋白质组学和质谱设备中进行。吉布森博士是该设施的负责人，该设施能够对蛋白质的复杂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的复合物进行详细分析。吉布森博士也将在爱荷华州的核磁共振设备工作。他们将从CLM制剂中获取1维和2维NMR数据