A streamlined platform for phosphoproteome mapping of human tissues

人体组织磷酸化蛋白质组图谱的简化平台

• 批准号: 10687348
• 负责人: Huiping Liu
• 金额: $ 60万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-09 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10687348
• 关键词: 3-Dimensional; Address; Affinity Chromatography; Antibodies; Antibody Specificity; Biology; Breast; Cell physiology; Cells; Collection; Communities; Complex; Coupled; DNA; Data; Detection; Development; Dimensions; Disease; Generations; Genomics; Goals; Health; Heterogeneity; Human; Human BioMolecular Atlas Program; Human body; Image; Immobilization; Immunoassay; Ions; Knowledge; Label; Link; Liquid Chromatography; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Metals; Methods; Modification; Movement; Mus; Performance; Phase; Phosphopeptides; Phosphorylation; Preparation; Proteins; Proteome; Proteomics; RNA; Recovery; Research; Resolution; Sampling; Signal Pathway; Signal Transduction; Slide; Source; Specificity; Technology; Thick; Tissues; Tube; Uterus; Visualization software; base; cell type; detection sensitivity; human tissue; improved; in vivo; insight; instrument; ionization; laser capture microdissection; liquid chromatography mass spectrometry; nano-electrospray; open source; phosphoproteomics; precision medicine; pressure; single cell technology; stoichiometry; surfactant; technology development; tissue mapping; tool; transcriptomics

ABSTRACT Recent technological advances in genomics, transcriptomics, and proteomics allow for rapid generation of comprehensive 3D-human tissue maps for biomolecules DNAs, RNAs, and proteins at the single-cell resolution in the HuBMAP consortium. However, single-cell technologies for characterizing functional modifications are lagging far behind but equally important as these existing omics technologies. Protein phosphorylation is one of the most important modifications and often used as an indicator of signaling pathway activation (cell functional state). The lack of high-spatial-resolution phosphoproteomic characterization of human tissues in the HuBMAP consortium represents a significant knowledge gap for achieving a more complete understanding of how tissue heterogeneity impacts human health. The objective of this TTD application is to address this gap by developing a convenient streamlined platform for enabling automated high-resolution 3D-phosphoproteome mapping of human tissues. The project feasibility is strongly supported by our recent progress in many aspects of technology development: 1) Carrier-assisted sample preparation (CASP) for both global and targeted proteomics analysis of 1-100 cells; 2) A boosting to amplify signal with isobaric labeling (BASIL) strategy for high-throughput single- cell proteomics; 3) BASIL/Tip-IMAC (immobilized metal affinity chromatography) for rapid phosphoproteomic analysis of small numbers of cells; 4) Advanced liquid chromatography (LC) separation and disruptive mass spectrometry (MS) technologies (e.g., multi-emitter array technology and sub-ambient pressure ionization with nanoelectrospray (SPIN) source) for improving MS detection sensitivity. In the UG3 phase, Aim 1 will focus on the development of a streamlined platform through 1) improving phospho-recovery by developing an improved CASP/online IMAC platform for automated processing and phospho-enrichment, and 2) leveraging multiple disruptive technologies developed at our group with integration of a high-efficiency multi-emitter SPIN (mSPIN) source and BASIL-based sample multiplexing for significantly improving MS sensitivity by ~50-fold and sample throughput by >20-fold. The streamlined platform will allow for precise quantification of ~1,000 phosphosites in single cells and ~7000 phosphosites in 10 cells with >1000 samples per day. Aim 2 will demonstrate the streamlined platform for enabling 2D-phosphoproteome mapping of mouse uterine tissues when combined with laser capture microdissection (LCM) and standard tube-based voxel collection. In the UH3 phase (Aim 3) we will further optimize the streamlined platform for automated robust phosphoproteomic analysis of LCM-dissected human tissue sections. We then will validate the streamlined platform for high-resolution 3D-phosphoproteome mapping of human breast and uterine tissues and other human tissues from the HuBMAP consortium. An easy- to-use visualization tool will be developed to generate 3D maps that can be quickly and easily accessible by the research community. With its antibody-free feature, the streamlined platform can be equally applicable to any types of tissues. We envision that the streamlined platform will become an indispensable tool for high-resolution 3D-phosphopeoteome mapping of human tissues in the HuBMAP consortium and extend the HuBMAP toolbox for 3D-mapping of functional modifications. In turn, it will make substantial contributions to improve our understanding of tissue biology and accelerate the movement toward precision medicine.

抽象的 基因组学，转录组学和蛋白质组学的最新技术进步允许快速生成 单细胞分辨率的生物分子DNA，RNA和蛋白质的综合3D人类组织图 在Hubmap联盟中。但是，用于表征功能修改的单细胞技术是 与这些现有的OMICS技术一样落后，但同样重要。蛋白质磷酸化是 最重要的修改，通常用作信号通路激活的指标（细胞功能 状态）。人体组织中缺乏高空间分辨率的磷酸化表征 财团代表了一个显着的知识差距，以实现对组织的更全面了解 异质性影响人类健康。该TTD应用程序的目的是通过开发来解决这一差距 一个方便的简化平台，用于实现自动高分辨率3D-磷蛋白质组映射 人体组织。我们最近在技术的许多方面的进步得到了强烈支持该项目的可行性 开发：1）载体辅助样品制备（CASP），用于全球和靶向蛋白质组学分析 1-100个细胞； 2）通过等速标记（罗勒）策略来扩大信号的增强信号，以进行高通量单个 细胞蛋白质组学； 3）basil/tip-imac（固定的金属亲和力色谱法），用于快速磷酸蛋白质组学 分析少量细胞； 4）晚期液相色谱（LC）分离和破坏性质量 光谱法（MS）技术（例如，多发射极阵列技术和次级型压力电离与 用于提高MS检测灵敏度的纳米电喷雾（自旋）源。在UG3阶段，AIM 1将重点放在 通过1）通过改进的改进来开发流线型平台1） CASP/在线iMac平台用于自动加工和磷酸化富集，以及2）利用多个 在我们小组中开发的破坏性技术，并结合了高效的多发旋体旋转（MSPIN） 源和基于罗勒的样品多路复用，以显着提高MS灵敏度，并将样品提高约50倍和样品 吞吐量> 20倍。简化的平台将允许精确量化约1,000个磷石 在单个细胞中，在10个细胞中，每天有> 1000个样品中的磷酸材料。 AIM 2将证明 当与 激光捕获微分解（LCM）和基于标准管的体素收集。在UH3阶段（目标3）我们将 进一步优化流线型平台，以进行自动化的LCM触发磷光蛋白质组学分析 人体组织切片。然后，我们将验证用于高分辨率3D-磷蛋白质组的流线型平台 人类乳房和子宫组织和其他人类组织的映射。一个简单的 将开发出使用的可视化工具来生成3D地图，可以快速，易于访问 研究社区。具有无抗体功能，流线型平台同样适用于任何 组织类型。我们设想流线型平台将成为高分辨率的必不可少的工具 Hubmap财团中人体组织的3D磷化蛋白体映射并扩展了Hubmap工具箱 用于功能修改的3D映射。反过来，这将为改善我们的贡献做出重大贡献 了解组织生物学并加速迈向精确医学的运动。