Microparticle Capture-Based Analysis of Lymph Node Proteome

基于微粒捕获的淋巴结蛋白质组分析

• 批准号: 8650786
• 负责人: Virginia Anne Espina
• 金额: $ 22.09万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-15 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8650786
• 关键词: Acute; Address; Affinity; Animals; Anthrax disease; Antibiotics; Architecture; Bacillus anthracis; Bacillus anthracis spore; Bacterial Proteins; Bacterial Toxins; Biological Assay; Blood Vessels; Carbon Dioxide; Chemistry; Communicable Diseases; Complement; Complex; Cytolysis; Data; Development; Disease; Dyes; Environment; Extravasation; Functional disorder; Genes; Global Change; Goals; Harvest; Hemolysin; Hemorrhage; Hour; Hydrogels; Hypoxia; Immune; Immunoassay; Immunosuppression; In Situ; Infection; Inflammatory Response; Knowledge; Lasers; Location; Lymph; Lymph Node Sinus; Lymph Node Tissue; Lymphadenitis; Lymphatic; Lymphatic System; Mass Spectrum Analysis; Mediator of activation protein; Medical; Metabolic; Microarray Analysis; Mitochondria; Modeling; Molecular; Molecular Weight; Mus; One-Step dentin bonding system; Organ; Oxygen; Pathogenesis; Pathogenicity; Peptide Hydrolases; Phase; Preparation; Procedures; Process; Property; Prophylactic treatment; Protein Microchips; Proteins; Proteome; Proteomics; Reproduction spores; Research; Rodent; Rodent Model; Role; Sample Size; Sampling; Sentinel Lymph Node; Series; Serum; Spleen; Techniques; Technology; Time; TimeLine; Tissues; Toxin; Tracer; Virulence Factors; Yersinia pestis; anthrax toxin; base; cell injury; direct application; effective therapy; in vivo; inhibitor/antagonist; laser capture microdissection; lymph nodes; macrophage; mutant; nanoparticle; novel; pathogen; public health relevance; research study; tool

DESCRIPTION (provided by applicant): Lymphadenitis takes place in the pathogenesis of many infectious diseases, including the ones caused by the bioterror agents such as Y. pestis, F. tularenis, and B. anthracis. In the case of B. anthracis, the lymphatic system serves as the conduit by which germinating spores are delivered by macrophages to the sentinel lymph nodes (SLNs) within hours after exposure. The critical role of SLNs in anthrax makes them important targets for proteomic analyses. For this purpose, we will develop a novel nanoparticle-based technique to study the dynamics of SLN proteome during infection. Currently, direct application of the proteomic tools to study HL has a number of limitations relevant to the assay sensitivity, sample size and preparation procedures, especially in small-rodent models often used in anthrax research. To alleviate these limitations we invented the core-shell, affinity bait, hydroge nanoparticles that quickly capture, protect from degradation and separate from the abundant proteins the low-molecular-weight (LMW) fraction of the sample proteins in one step for direct downstream analysis by mass spectrometry (MS). We will identify the host protein targets associated with tissue damage and obtain new information on the hemorrhage-inducing mechanism of pathogenicity operating in the specific environment of lymphatics. At certain time points during the course of infection the SLNs will be visualized with the tracer dye. We will inject SLNs with harvesting nanoparticles to capture biomolecules within the SLN interstitium for deciphering its proteomic composition. Nanoparticles will be readily retrieved from the surgically excised SLNs by laser capture microdissection of the whole lymph node or any portion of its architecture. The nanoparticle-harvested biomolecules can be further analyzed by any analytical platform including MS, immunoassays, or microarrays. Our preliminary data support feasibility of the suggested experimental approach. The core shell nanoparticles containing different baits demonstrate high-affinity capture of host and bacterial proteins that can be reliably identified by MS and further validated using the highly-sensitive, high-throughput proteomics platform, Reverse-Phase Microarray (RPMA). The approach we develop will be broadly applicable to analyses of SLNs during different diseases. Aim 1. Inject nanoparticles into lymph nodes in a murine model to harvest the in vivo proteome of the LN microenvironment. Aim 2: Assess the LN proteome during the time course of anthrax infection with lethal and non-lethal strains.

描述（由申请人提供）：淋巴结炎发生在许多感染性疾病的发病机制中，包括由生物恐怖剂如Y。pestis，F. tularenis和B.炭疽病在B的情况下。在炭疽病中，淋巴系统充当管道，通过该管道，在暴露后数小时内，巨噬细胞将萌发的孢子递送到前哨淋巴结（SLN）。SLN在炭疽中的关键作用使其成为蛋白质组学分析的重要目标。为此，我们将开发一种新的基于纳米颗粒的技术来研究感染过程中SLN蛋白质组的动态变化。目前，直接应用蛋白质组学工具来研究HL具有与测定灵敏度、样品大小和制备程序相关的许多限制，特别是在炭疽研究中经常使用的小型啮齿动物模型中。为了减轻这些限制，我们发明了核-壳、亲和诱饵、水纳米颗粒，其在一个步骤中快速捕获样品蛋白质的低分子量（LMW）级分，防止降解并从丰富的蛋白质中分离，用于通过质谱（MS）进行直接下游分析。我们将确定与组织损伤相关的宿主蛋白质靶点，并获得在特定环境下致病性的损伤诱导机制的新信息。在感染过程中的某些时间点，将用示踪染料观察SLN。我们将向SLN注射收获纳米颗粒，以捕获SLN内的生物分子，从而破译其蛋白质组组成。通过对整个淋巴结或其结构的任何部分进行激光捕获显微解剖，将容易地从手术切除的SLN中回收纳米颗粒。纳米颗粒收获的生物分子可以通过任何分析平台进一步分析，包括MS、免疫测定或微阵列。我们的初步数据支持建议的实验方法的可行性。含有不同诱饵的核壳纳米颗粒表现出对宿主和细菌蛋白质的高亲和力捕获，这些蛋白质可以通过 MS，并使用高灵敏度，高通量蛋白质组学平台，反相微阵列（RPMA）进一步验证。我们开发的方法将广泛适用于分析不同疾病的前哨淋巴结。 目标1.将纳米颗粒注射到小鼠模型的淋巴结中以收获LN微环境的体内蛋白质组。 目的2：研究炭疽致死株和非致死株感染过程中LN蛋白质组的变化。