Mass Spectrometric Characterization of Lipopolysaccharides

描述（由申请人提供）：革兰氏阴性菌是世界上一些最致命和更广泛的流行病的原因。幽门螺杆菌（Helicobacter pylori，Hp）是引起消化性溃疡和胃癌的主要致病菌，空肠弯曲杆菌（Camporbacter jejuni，空肠弯曲菌）和霍乱弧菌（Vibrio cholera，霍乱弧菌）是引起消化性溃疡和胃癌的主要致病菌，鲍曼不动杆菌（Acinetobacter baumannii，鲍曼不动杆菌）是引起医院内耐药菌感染的主要致病菌。了解这些细菌用于启动发病机制，识别和激活免疫系统以及发展抗生素耐药性的机制是需要多学科研究策略的重要问题。拟议的工作重点是开发先进的质谱方法，用于表征复杂的脂多糖（LPS），包括内毒素脂质A亚单位，包括革兰氏阴性菌外膜的关键成分。考虑到LPS和特别是脂质A结构中所见的多样性，LPS的结构表征是一项具有挑战性的任务。我们已经开始发展三种光解离方法，包括红外多光子解离（IRMPD），紫外光解离（UVPD），和活化电子光解离解离（a-EPD），用于表征脂质A和LPS的结构。具体目标包括：1）用于表征脂质A和核心寡糖/O-抗原的光解离和混合MS/MS方法。第一个目标需要系统检查的裂解模式获得的光解离和混合MS/MS方法的脂质A和寡糖化合物。2)LPS的自上而下表征。完整LPS的表征将通过将自下而上方法（脂质和寡糖）获得的片段化模式与通过中间向下策略从越来越大的脂多糖部分获得的信息相结合，然后进行到集成的自上而下工作流程。3)计算机数据库搜索算法的开发。MS/MS光谱的复杂性（包含来自脂质和糖部分的碎片离子阵列）使其解释具有挑战性。合作者Hua Xu将开发一种计算机数据库搜索算法MassMatrixLPS，以促进自动化，高通量数据分析。4)应用于H. pylori、C. jejuni、霍乱弧菌和E.杆菌Brodbelt和Trent小组之间的合作旨在阐明三种变形菌的LPS修饰系统，并阐述它们的结构/功能关系。具体的生物学问题包括脂质A的结构变化与炎症反应的相关性，阐明LPS的生物合成途径，以及阐明抗菌肽的耐药性机制。 公共卫生相关性：革兰氏阴性菌是世界上一些最致命和更广泛的流行病的罪魁祸首。拟议的工作重点是开发先进的质谱方法来表征复杂的脂多糖，包括内毒素脂质A结构域，包括革兰氏阴性菌的关键外膜。

DESCRIPTION (provided by applicant): Gram-negative bacteria are responsible for some of the deadliest and more widespread pandemics in the world. Helicobacter pylori is now recognized as the primary cause of peptic ulcer disease and gastric cancer, Camphorbacter jejuni and Vibrio cholera remain among the most common causes of diarrheal illness, and Acinetobacter baumannii is responsible for a growing number of the antibiotic-resistant infections in hospitals. Understanding the machinery of these bacteria used for initiation of pathogenesis, for recognition and activation of the immune system, and for development of antibiotic resistance are vital issues that require multi-disciplinary research strategies. The proposed work focuses on the development of advanced mass spectrometric approaches for characterization of the complex lipopolysaccharides (LPS), including the endotoxic lipid A sub-unit, that comprise the key constituents of the outer membrane of Gram-negative bacteria. Given the diversity seen in LPS and particularly lipid A structures, the structural characterizatio of LPS is a challenging task. We have begun to develop three photodissociation methods, including infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), and activated-electron photodetachment dissociation (a-EPD), for the characterization of lipid A and LPS structures. Specific objectives include: 1) Photodissociation and hybrid MS/MS methods for characterization of lipid A and core oligosaccharide/O-antigens. The first aim entails systematic examination of the fragmentation patterns obtained by photodissociation and hybrid MS/MS methods for lipid A and oligosaccharide compounds. 2) Top-down characterization of LPS. Characterization of intact LPS will build by combining the fragmentation patterns obtained from bottom-up approaches (lipids and oligosaccharides) with information obtained from increasingly larger portions of the lipopolysaccharides via a middle-down strategy, then progressing to an integrated top-down workflow. 3) Development of an in silico database search algorithm. The complexity of the MS/MS spectra which contain an array of fragment ions from both the lipid and sugar portions makes their interpretation challenging. An in silico database search algorithm, MassMatrixLPS, will be developed by collaborator Hua Xu to facilitate automated, higher-throughput data analysis. 4) Applications to lipid A and LPS of H. pylori, C. jejuni, V. cholera, and E. coli. The collaboration forged between the Brodbelt and Trent groups is aimed at elucidating the LPS modification systems of three proteobacteria and elaborating their structure/function relationships. Specific biological problems include correlation of the structura changes of lipid A with the inflammatory response, unraveling the biosynthetic pathway of LPS, and elucidating the mechanism of resistance to antimicrobial peptides. PUBLIC HEALTH RELEVANCE: Gram-negative bacteria are responsible for some of the deadliest and more widespread pandemics in the world. The proposed work focuses on the development of advanced mass spectrometric approaches for characterization of the complex lipopolysaccharides, including the endotoxic lipid A domains that comprise the key outer membrane of Gram-negative bacteria.