Proteomics Pathway Discovery

由病毒感染触发，PRR介导的免疫应答途径是高度稳健的， 复杂的涉及多个，瞬时组装的蛋白质复合物，在一个 相互关联和表型的方式。这种复杂的组装和多样化的途径需要新的 技术能够系统地分析这些关键途径的组成部分， 精确的方法。我们之前已经证明了我们发现蛋白质组学的独特优势 用于识别/分析新途径成分的平台，包括那些动态蛋白质-蛋白质 相互作用（PPI）和翻译后修饰（PTM）参与调控的起始， PRR介导的免疫信号传导的进展和终止。我们还表明，这些结果从 对PRR介导的通路进行蛋白质组学分析可以指导和简化深入的功能分析 使用传统的遗传学、细胞生物学和免疫学方法，从而扩大了 对宿主免疫反应的机械理解。凭借这一丰富的经验， 该共享资源核心B的目的是枚举后续信令的组成部分 由先天免疫PRR与病毒相互作用引起的途径。所有定量蛋白质组学分析 PPI和PTM的两个主要途径操作者，以及病毒诱导的蛋白质易位， 对感染受试者与未感染对照或不同感染阶段进行比较， 无论是全球或亚细胞区室或在细胞器特异性的方式。各种量化方案 包括AACT/SILAC、ITRAQ和无标记定量将在我们的实验中补充使用 不仅用于细胞系，而且用于原代细胞或组织。此外，我们将积极参与指导 所有项目的样品制备，特别是与新开发技术相关的项目， 蛋白质组学结果将产生多个假设（即发现新的蛋白质，PPI或PTM）， 这两种相互关联和有效的方式，以指导同时发现和阐明新的 生物学、途径和机制。特别是通过应用最先进的定量技术 蛋白质组学，我们将（1）剖析全球或特定路径的蛋白质-蛋白质相互作用网络 （相互作用组）参与PRR介导的病毒感染免疫反应，以及（2）识别/分析全球 或相互作用组特异性翻译后修饰（PTM），包括磷酸化、乙酰化和 泛素化参与了PRR介导的对病毒感染的免疫应答。

Triggered by viral infection, the PRR-mediated immune response pathways are highly robust and complicated with the involvement of multiple, transiently assembled protein complexes that function in an interconnected and phenotypic manner. This complex assembly and diverse pathways demand new technology capable of systematically profiling those critical pathway components in more sensitive and precise approaches. We have previously demonstrated the unique strength of our discovery proteomics platform in identifying/profiling novel pathway constituents, including those dynamic protein-protein interactions (PPIs) and post-translational modifications (PTMs) involved in the regulation of the initiation, progression and termination of PRR-mediated immune signaling. We also showed that these results from the proteomic dissection of PRR-mediated pathways could guide and streamline in-depth functional analyses using conventional genetic, cell biology, and immunology approaches, thereby enlarging the scope of mechanistic understanding of the host immune response. Leveraging this extensive experience, the overall objective of this shared resource Core B is to enumerate the constituents of the subsequent signaling pathways caused by the interaction of innate immune PRRs with viruses. All quantitative proteomic analyses of PPIs and PTMs, the two major pathway operators, as well as virus-induced protein translocations, can be performed comparatively for infected subjects versus non-infected controls or at different infection stages in either global or subcellular compartments or in organelle-specific fashion. Various quantitative schemes including AACT/SILAC, ITRAQ, and label-free quantitation will be complimentarily used in our experiments for not only cell lines, but also primary cells or tissues. Additionally, we will be significantly involved in guiding sample preparation in all Projects, particularly those associated with newly developed techniques of proteomics. Results will generate multiple hypotheses (i.e. discovery of novel proteins, PPIs or PTMs) in both interconnected and efficient manners to guide concurrently the discovery and elucidation of novel biologies, pathways and mechanisms. Specifically by applying the state-of-the-art technology of quantitative proteomics, we will (1) dissect either global or pathway-specific protein-protein interaction networks (interactome) involved in the PRR-mediated immune response to viral infection, and (2) identify/profile global or interactome-specific post-translational modifications (PTMs) including phosphorylation, acetylation, and ubiquitination involved in the PRR-mediated immune response to viral infection.