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: 10707474
• 负责人: Huiping Liu
• 金额: $ 57.85万
• 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/10707474
• 关键词: 3-Dimensional; Acceleration; 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; cell type; detection sensitivity; human tissue; improved; in vivo; insight; instrument; ionization; laser capture microdissection; liquid chromatography mass spectrometry; meter; 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使得能够快速生成 单细胞分辨率的生物分子 DNA、RNA 和蛋白质的综合 3D 人体组织图 在 HuBMAP 联盟中。然而，用于表征功能修饰的单细胞技术是 远远落后但与这些现有的组学技术同样重要。蛋白质磷酸化是其中之一 最重要的修饰，通常用作信号通路激活（细胞功能 状态）。 HuBMAP 中缺乏人体组织的高空间分辨率磷酸化蛋白质组学特征 联盟代表了一个重大的知识差距，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来弥补这一差距 一个方便的简化平台，用于实现自动化高分辨率 3D 磷酸化蛋白质组图谱 人体组织。该项目的可行性得到了我们近期在多方面技术进展的有力支持 开发：1) 载体辅助样品制备 (CASP)，用于全局和靶向蛋白质组学分析 1-100个细胞； 2) 采用同量异位标记 (BASIL) 策略增强信号放大，用于高通量单 细胞蛋白质组学； 3) BASIL/Tip-IMAC（固定化金属亲和层析）用于快速磷酸化蛋白质组学 分析少量细胞； 4) 先进的液相色谱 (LC) 分离和破坏质量 光谱测定 (MS) 技术（例如，多发射器阵列技术和亚环境压力电离技术） 纳米电喷雾 (SPIN) 源），用于提高 MS 检测灵敏度。在UG3阶段，目标1将重点关注 通过以下方式开发简化的平台： 1) 通过开发改进的方法来提高磷回收率 用于自动处理和磷酸富集的 CASP/在线 IMAC 平台，以及 2) 利用多个 我们团队开发的颠覆性技术，集成了高效多发射器 SPIN (mSPIN) 源和基于 BASIL 的样品多重分析可将 MS 灵敏度显着提高约 50 倍，并且样品 吞吐量提高了 20 倍以上。简化的平台将允许精确定量约 1,000 个磷酸位点 单细胞中含有约 7000 个磷酸位点，10 个细胞中含有约 7000 个磷酸位点，每天有超过 1000 个样本。目标 2 将展示 简化的平台，结合使用可实现小鼠子宫组织的 2D 磷酸化蛋白质组图谱 激光捕获显微切割 (LCM) 和标准管基体素采集。在 UH3 阶段（目标 3），我们将 进一步优化简化的平台，用于 LCM 解剖的自动稳健磷酸蛋白质组分析 人体组织切片。然后，我们将验证高分辨率 3D 磷酸蛋白质组的简化平台 HuBMAP 联盟绘制的人类乳房和子宫组织以及其他人体组织的图谱。一个简单的—— 将开发易用的可视化工具来生成 3D 地图，用户可以快速轻松地访问这些地图 研究社区。凭借其无抗体的特点，简化的平台可以同样适用于任何 组织类型。我们预计，精简的平台将成为高分辨率的不可或缺的工具。 HuBMAP 联盟中人体组织的 3D 磷酸化组图谱并扩展 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 了解组织生物学并加速精准医学的发展。