Microparticle Capture-Based Analysis of Lymph Node Proteome

描述（由申请人提供）：淋巴结炎发生在许多传染病的发病机制中，包括由生物恐怖剂如鼠疫杆菌、土拉菌和炭疽杆菌引起的疾病。在炭疽芽胞杆菌的情况下，淋巴系统作为管道，在暴露后数小时内，巨噬细胞将萌发的孢子传递到前哨淋巴结（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粒子的技术来研究感染过程中SLN蛋白质组的动力学。目前，直接应用蛋白质组学工具来研究HL存在许多与检测灵敏度、样本量和制备程序相关的局限性，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、氢纳米颗粒，这些纳米颗粒可以快速捕获、防止降解并从丰富的蛋白质中分离出样品蛋白质的低分子量（LMW）部分，一步即可通过质谱（MS）直接进行下游分析。我们将确定与组织损伤相关的宿主蛋白靶点，并获得在淋巴特定环境中运作的致病性诱导出血机制的新信息。在感染过程中的某些时间点，示踪染料将显示sln。我们将在SLN中注入纳米颗粒，以捕获SLN间质内的生物分子，从而破译其蛋白质组学组成。通过激光捕获整个淋巴结或其结构的任何部分的显微解剖，可以很容易地从手术切除的sln中提取纳米颗粒。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，包括质谱、免疫测定或微阵列。我们的初步数据支持所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，可以可靠地识别

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.