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.

摘要 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和蛋白质的全面三维人体组织图 在HuBMAP联盟。然而，用于表征功能性修饰的单细胞技术是不成熟的。 远远落后于这些现有的组学技术，但同样重要。蛋白质磷酸化是 最重要的修饰，并经常用作信号通路激活的指标（细胞功能 州）。HuBMAP中缺乏人体组织的高空间分辨率磷酸化蛋白质组学表征 联盟代表了一个重大的知识差距，以实现更完整的了解组织如何 异质性影响人类健康。本TTD应用程序的目标是通过开发 一个方便的流线型平台，用于实现自动化的高分辨率3D磷酸化蛋白质组图谱， 人体组织项目的可行性得到了我们最近在许多技术方面取得的进展的有力支持 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）用于高通量单克隆抗体的加强以用同量异位素标记（BASIL）策略放大信号; 细胞蛋白质组学; 3）用于快速磷酸化蛋白质组学的BASIL/Tip-IMAC（固定化金属亲和层析） 分析少量细胞; 4）先进的液相色谱（LC）分离和破碎质量 光谱分析（MS）技术（例如，多发射器阵列技术和亚环境压力电离， 纳米电喷雾（SPIN）源）用于提高MS检测灵敏度。在UG 3阶段，目标1将侧重于 通过1）通过开发改进的 CASP/在线IMAC平台，用于自动化处理和磷酸富集，以及2）利用多个 我们团队开发的突破性技术，集成了高效多发射极SPIN（mSPIN） 源和基于BASIL样品复用，可将MS灵敏度显著提高约50倍， 吞吐量增加20倍以上。精简的平台将允许精确定量约1，000个磷酸化位点 在单个细胞中，在10个细胞中有约7000个磷酸化位点，每天有>1000个样品。目标2将展示 当与以下组合时，用于实现小鼠子宫组织的2D-磷酸化蛋白质组图谱的流线型平台 激光捕获显微切割（LCM）和标准的基于管的体素收集。在UH 3阶段（目标3），我们将 进一步优化流线型平台，用于LCM切割的自动化稳健磷酸化蛋白质组学分析， 人体组织切片然后，我们将验证高分辨率3D磷酸化蛋白质组的简化平台 人类乳腺和子宫组织以及来自HuBMAP联盟的其他人类组织的图谱。简单的- 将开发一个使用可视化工具来生成3D地图， 研究社区。凭借其无抗体功能，流线型平台同样适用于任何 组织的类型。我们设想，精简的平台将成为一个不可或缺的工具， HuBMAP联盟中人体组织的3D磷酸酶组图谱，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 组织生物学的理解，并加速向精准医学的发展。