BioAnalytical Chemistry Core B: Innate reponses to microbial infection

生物分析化学核心B将为该计划项目拨款的三个项目提供专业的生物分析化学支持，即对微生物感染的先天反应。这些区域包括质量 光谱学、蛋白质化学与蛋白质组学(第一部分)、碳水化合物与脂肪化学(第二部分)。 在第一部分中，将对人中性粒细胞制剂-胶囊状材料(CLM)进行蛋白质组学比较分析，包括基于发现的蛋白质组学方法和基于定量质谱学的蛋白质组学方法。 和各种感兴趣的目标蛋白。项目1(Apicella)。我们将检查可能存在脂蛋白锚和的CLM补偿。如果存在，它的特性和脂类修饰将被阐明。 作为项目2(Nauseef)的一部分。我们将针对对调节至关重要的人类细胞类型或 先天免疫反应的失调，并确定耐洗涤剂膜微域或脂筏中包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(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，将使用稳定同位素标记多肽的多反应监测方法进行有针对性的定量。 在第二部分中，我们将使用我们在质谱学和碳水化合物和脂类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。与Apicella博士(项目1)一起，我们将对CLM制剂进行详尽的分析，以确定其多糖结构和膜锚，可能是二酰甘油磷酸连接的部分。项目2(Nauseef)。 将对招募的中性粒细胞中磷脂成分的变化进行检查。 所有实验都将在巴克研究所的蛋白质组学和质谱学设施中进行。该设施由Gibson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肪的复合体进行详细分析。吉布森博士还将与爱荷华州的核磁共振设施合作。世卫组织将从CLM制剂中获取1和2D核磁共振数据

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