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.

摘要 基因组学、转录组学和蛋白质组学的最新技术进步使快速产生 以单细胞分辨率显示生物分子DNA、RNA和蛋白质的全面3D人体组织图 在HuBMAP财团中。然而，用于表征功能修饰的单细胞技术是 远远落后于这些现有的组学技术，但与它们同等重要。蛋白质磷酸化是一种 最重要的修饰，通常用作信号通路激活的指标(细胞功能 州)。HuBMAP缺乏高空间分辨率的人体组织磷蛋白质组学特征 该联盟代表着一项重大的知识鸿沟，无法更全面地了解组织如何 异质性影响人类健康。此TTD应用程序的目标是通过开发 一个方便、简化的平台，用于实现自动化的高分辨率三维磷酸蛋白质组图谱 人体组织。我们最近在许多方面的技术进步有力地支持了该项目的可行性 发展：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)等压标记增强信号放大(BASIL)策略，用于高通量单细胞 细胞蛋白质组学；3)Basil/Tip-iMac(固定化金属亲和层析)快速磷酸蛋白质组学 少量细胞的分析；4)先进的液相色谱(LC)分离和破坏性物质 光谱(MS)技术(例如，多发射极阵列技术和亚常压电离 纳米电喷雾(SPIN)源)，以提高MS检测灵敏度。在UG3阶段，目标1将专注于 流线型平台的开发通过1)通过开发改进的 用于自动化处理和磷浓缩的CASP/在线iMac平台，以及2)利用多个 我们集团开发的颠覆性技术集成了高效多发射器自旋(MSPIN) 基于源和罗勒的样品多路复用显著提高MS灵敏度约50倍和样品 吞吐量增加了20倍。流线型平台将允许精确量化~1,000个亚磷酸盐 单细胞和10个细胞中约7000个亚磷酸盐，每天1000个样本。目标2将演示 当结合使用时，支持小鼠子宫组织2D-磷酸蛋白质组图谱的流线型平台 激光捕获显微解剖(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LCM-解剖自动健壮磷酸蛋白质组分析的优化平台 人体组织切片。然后，我们将验证用于高分辨率3D-磷酸蛋白质组的流线型平台 来自HuBMAP联盟的人类乳房和子宫组织以及其他人类组织的地图绘制。一个简单的- 将开发可使用的可视化工具来生成3D地图，这些地图可以快速轻松地通过 研究社区。由于其无抗体功能，简化的平台可以同样适用于任何 纸巾的种类。我们设想，精简的平台将成为高分辨率的不可或缺的工具 在HuBMAP联盟中对人体组织进行3D-磷酸组图谱绘制并扩展HuBMAP工具箱 用于功能修改的3D映射。反过来，它将为改善我们的 了解组织生物学，加速向精准医学迈进。