Mass Spectrometric Characterization of Lipopolysaccharides

脂多糖的质谱表征

• 批准号: 8547083
• 负责人: Jennifer S. Brodbelt
• 金额: $ 26.85万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-18 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8547083
• 关键词: Acinetobacter baumannii; Advanced Development; Algorithms; Anti-Inflammatory Agents; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Bacteria; Biological; Campylobacter jejuni; Cell membrane; Cells; Collaborations; Complex; Computer Simulation; Cytolysis; Data Analyses; Databases; Development; Disease; Dissociation; Electrons; Environment; Escherichia coli; Exhibits; Gene Deletion; Goals; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Guillain-Barré Syndrome; Helicobacter pylori; Helicobacter pylori lipopolysaccharide; Hospitals; Human; Hybrids; Immune; Immune system; Infection; Inflammatory Response; Ions; Lead; Lipid A; Lipids; Lipopolysaccharides; Membrane; Methods; Modification; Mutation; Neisseria meningitidis; O Antigens; Oligosaccharides; Organism; Pathogenesis; Pathway interactions; Pattern; Peptic Ulcer; Polysaccharides; Proteins; Proteobacteria; Reactive Arthritis; Research; Resistance; Salmonella; Serum; Stomach; Structure; Structure-Activity Relationship; Surface; System; Systems Development; Toll-like receptors; Vaccines; Variant; Vibrio cholerae; Work; Yersinia pestis; antimicrobial peptide; design; forging; interest; lead-binding proteins; malignant stomach neoplasm; microbial; pandemic disease; pathogenic Escherichia coli; sugar; ultraviolet

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.

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罪魁祸首。幽门螺杆菌现在被认为是消化性溃疡疾病和胃癌的主要原因，空肠Camphorbacter空肠弧菌和霍乱弧菌仍然是腹泻疾病的最常见原因，鲍曼不动杆菌是医院中越来越多的抗生素耐药感染的原因。了解这些用于启动发病机制、识别和激活免疫系统以及抗生素耐药性发展的细菌机制是需要多学科研究策略的重要问题。提出的工作重点是发展先进的质谱方法来表征复杂的脂多糖（LPS），包括内毒素脂质A亚基，构成革兰氏阴性菌外膜的关键成分。鉴于LPS特别是脂质A结构的多样性，LPS的结构表征是一项具有挑战性的任务。我们已经开始开发三种光解方法，包括红外多光子解离（IRMPD），紫外光解离（UVPD）和活化电子光解离（A - epd），用于表征脂质A和LPS的结构。具体目标包括：1)光解和杂交MS/MS方法表征脂质A和核心寡糖/ o抗原。第一个目标是系统地检查通过光解和混合质谱/质谱方法获得的脂质A和低聚糖化合物的碎片模式。2)自上而下的LPS表征。完整LPS的表征将通过将自下而上方法（脂质和低聚糖）获得的碎片模式与通过中向下策略从越来越大的脂多糖部分获得的信息相结合，然后进展到一个集成的自上而下的工作流程。3)开发了一种计算机数据库搜索算法。复杂的质谱/质谱包含了一系列来自脂质和糖部分的碎片离子，这使得它们的解释具有挑战性。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，更高吞吐量的数据分析。4)幽门螺杆菌、空肠梭菌、霍乱弧菌和大肠杆菌脂质A和LPS的研究。broadbelt和Trent团队之间的合作旨在阐明三种变形菌的LPS修饰系统，并阐明它们的结构/功能关系。具体的生物学问题包括脂质A结构变化与炎症反应的相关性，揭示脂多糖的生物合成途径，阐明抗微生物肽的耐药机制。