TR&D 7: Cell Specific Proteomics

技术研发项目7：细胞特异性蛋白质组学 摘要 定量蛋白质组学的主要挑战之一是由于采样导致的信号稀释。因此在 在这个TR&D中，我们试图创建和完善三个工作流程，以在之前对10，000 - 100，000个特定类型的细胞进行采样， 通过自上而下的蛋白质组学（TDP）进行蛋白质组解析分析。我们制定了一个包括三个方面的攻击计划 临床样品的常见种类，即外周血单核细胞（PBMC）（DBP 5）、脑 切片（DBP 10、12和13）、病变组织（DBP 12和14）和实体瘤（DBP 9）。我们提出了三 实现细胞特异性和空间定位的主要策略，范围从可主动部署的荧光- 活化细胞分选（FACS），到更古老的激光捕获显微切割（LCM）方法，到一种新的， 更直接的方法，采用皮秒红外激光（PIRL）在自上而下之前消融完整的蛋白质 蛋白质组学随后，我们将通过我们的定量TDP平台分析收集的细胞特异性样品， 发现和靶向模式，后者利用已经开发的蛋白质型特异性测定（如 对于DBP 9中的KRAS蛋白形式）或在整个拟议的授予过程中从TR&D 6中出现的更多蛋白形式 期一个具体的例子来自我们对DBP 5（肾和肝移植患者）中PBMC的研究， 在那里发现了一组30种蛋白形式。从这些候选人中，我们现在需要 通过细胞类型分辨采样确定哪些免疫细胞对该信号最负责。 由此产生的蛋白质型分辨测量将提供更精确、更丰富的背景，因此是有价值的。 揭示蛋白质如何在人类疾病中发挥作用的数据。尽管小区特定的TDP技术目前不 存在，它的发展将通过大幅改善信息来解开蛋白质水平科学的重大瓶颈 关于蛋白质和细胞特异性的分子特征。虽然一些DBPs将受益于 在TDP之前通过FACS、LCM和PIRL对特定细胞和组织区域进行取样，这些技术 适用于各种生物背景下的蛋白质空间采样，这将直接受益于 在基础生物学，转化和临床研究的广泛研究。测量能力 还将扩大蛋白质组学的国家基础设施，帮助跟上竞争日益激烈的 国际科技格局。

Technology Research & Development Project 7: Cell Specific Proteomics Abstract One of the primary challenges of quantitative proteomics is the dilution of signal due to sampling. Therefore, in this TR&D, we seek to create and refine three workflows to sample 10,000-100,000 cells of specific types prior to proteoform-resolved analysis via top-down proteomics (TDP). We outline a plan of attack involving three common varieties of clinical samples, namely peripheral blood mononuclear cells (PBMCs) (DBP 5), brain sections (DBP 10, 12, and 13), diseased tissue (DBP 12 and 14) and solid tumors (DBP 9). We propose three main strategies to achieve cell specificity and spatial localization, range from actively deployable fluorescence- activated cell sorting (FACS), to the more venerable method of laser capture microdissection (LCM), to a novel, more direct method, which employs a picosecond infrared laser (PIRL) to ablate intact proteins prior to top-down proteomics. We will subsequently analyze collected cell-specific samples by our quantitative TDP platform in both discovery and targeted modes, with the latter utilizing proteoform-specific assays already developed (like for KRAS proteoforms in DBP 9) or the many more to emerge from TR&D 6 throughout the proposed granting period. A specific example derives from our work with PBMCs in DBP 5 (kidney and liver transplant patients), where a panel of 30 proteoforms has emerged from discovery work. From these candidates, we now need determine which immune cells are most responsible for this signal through cell type-resolved sampling. The resulting proteoform-resolved measurements will provide more precise, context-rich, and therefore valuable data revealing how proteins operate in human disease. Although cell-specific TDP technology does not presently exist, its development will unlock significant bottlenecks in protein-level science by sharply improved information about molecular signatures in a proteoform- and cell-specific fashion. While several DBPs will benefit from the sampling of specific cells and tissue regions prior to TDP by FACS, LCM, and PIRL, these technologies are applicable to the spatial sampling of proteins within a wide variety of biological contexts, which will directly benefit a broad range of studies in basic biological, translational and clinical research. The measurement capabilities will also augment the national infrastructure in proteomics, helping to keep pace with an increasingly competitive international landscape in science and technology.