Microparticle Capture-Based Analysis of Lymph Node Proteome

描述(申请人提供)：淋巴结炎在许多传染病的发病机制中发生，包括由生物恐怖因子如鼠疫杆菌、图拉氏杆菌和炭疽杆菌引起的疾病。就炭疽杆菌而言，淋巴系统是巨噬细胞在暴露后数小时内将萌发的孢子运送到前哨淋巴结(SLN)的管道。SLN在炭疽病中的关键作用使其成为蛋白质组学分析的重要靶标。为此，我们将开发一种新的基于纳米颗粒的技术来研究SLN蛋白质组在感染过程中的动态。目前，直接应用蛋白质组学工具来研究HL具有一定的局限性，尤其是在炭疽病研究中经常使用的小啮齿动物模型中，这与检测灵敏度、样本量和制备程序有关。为了缓解这些限制，我们发明了核壳、亲和诱饵和氢化纳米颗粒，这种纳米颗粒能够快速捕获、防止降解，并在一步内从丰富的蛋白质中分离出样品蛋白质的低分子量(LMW)部分，用于通过质谱仪(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，并获得关于在特定淋巴管环境中操作的致病机制的新信息。在感染过程中的特定时间点，将用示踪染料显示SLN。我们将向SLN注射收获的纳米颗粒，以捕获SLN间质中的生物分子，以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，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行进一步分析，包括MS、免疫分析或微阵列。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粒显示出对宿主和细菌蛋白的高亲和力捕获，可以通过 并使用高灵敏度、高通量的蛋白质组学平台--反相微阵列(RPMA)进一步验证。我们开发的方法将广泛适用于不同疾病期间SLN的分析。目的1.在小鼠淋巴结内注射纳米颗粒，以获取体内LN微环境的蛋白质组。目的：研究致死性和非致死性炭疽菌感染过程中LN蛋白质组的变化。

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.