Biodefense Proteomics Collaboratory

描述（由申请人提供）：将使用体外酶组合选择和分裂合成化学组合方法开发用于蛋白质组学的“硫代适体芯片”（TACh）-一种诊断工具，用于识别和定量响应生物恐怖主义威胁（BT）关注的病原体的关键蛋白质的差异表达。这项新的蛋白质组学技术将利用我们专有的硫代选择和硫代磷酸酯修饰的寡核苷酸“硫代适体”，结合我们的合作伙伴Ciphergen的表面增强激光解吸/电离（SELDI）质谱技术，以靶向啮齿动物和人类蛋白质组。特别地，我们将研究细胞因子和关键转录因子（例如，NF-κ B）。五个NF-？B/Rel家族蛋白可以联合收割机形成15种同源和异源二聚体，每一种在穿过细胞核膜易位并结合到基因的启动子区时执行特异性信号传导功能。在与Ciphergen的合作中，我们还将开发新的，大规模并行的，基于硫代适体珠的蛋白质组筛选与SELDI质谱方法，以确定参与对BT病毒的免疫反应的未表征的蛋白质。我们从TACh/SELDI方法的结果将通过2D凝胶质谱蛋白质组学方法进行验证。我们还将应用生物信息学分析将蛋白质表达的变化与病毒感染或休克后炎症引起的基因表达变化的可用基因组数据相关联。阐明这些蛋白质表达的变化将允许病毒性疾病的早期诊断和改善预后，以及随后开发有效的药理学和免疫学干预措施。特定的初始病毒靶点包括沙粒病毒、皮钦德病毒和拉沙病毒（后者在NIH和CDC A类名单上）。

DESCRIPTION (provided by applicant): In vitro enzymatic combinatorial selection and split-synthesis chemical combinatorial methods will be used to develop a "ThioAptamer Chip" (TACh) for proteomics - a diagnostic tool to identify and quantify the differential expression of key proteins in response to pathogens of concern for bioterrorism threat (BT). This new proteomics technology will utilize our proprietary thioselection and phosphorothioate-modified oligonucleotide "thioaptamers," combined with the surface enhanced laser desorption/ionization (SELDI) mass spectroscopy technology of our collaborating partner, Ciphergen, to target both rodent and human proteomes. In particular we will study the inflammatory response of cytokines and key transcription factors (e.g., NF-kappaB) challenged with BT agents. The five NF-?B/Rel family proteins can combine to form 15 homo- and heterodimers, each performing a specific signaling function upon translocation across the cell nuclear membrane and binding to a gene's promoter region. In partnership with Ciphergen, we will also develop new, massively parallel, thioaptamer bead-based screening of the proteome with SELDI mass-spectrometric methods to identify uncharacterized proteins involved in the immune response to BT viruses. Our results from the TACh/SELDI approaches will be validated by 2D gel mass spectrometric proteomic methods. We will also apply bioinformatic analyses to correlate changes in protein expression with available genomic data on changes in gene expression as a result of inflammation after viral infection or shock. Elucidating these protein expression changes will allow early diagnosis and enhanced prognosis of viral disease, and subsequent development of effective pharmacological and immunological interventions. Specific initial viral targets include arenaviruses, Pichinde and Lassa (the latter on both the NIH and CDC class A lists).