Renewable and Specific Affinity Reagents for Mapping Proteoforms in Human Tissues

用于绘制人体组织中蛋白质组图谱的可再生特异性亲和试剂

• 批准号: 9894469
• 负责人: NEIL L KELLEHER
• 金额: $ 59万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9894469
• 关键词: Affinity; Antibodies; Automation; Awareness; Bacteriophages; Biological; Cells; Cellular Structures; Chemicals; Colon; Colorectal; Confocal Microscopy; DNA; Data; Development; Epithelial Cells; Escherichia coli; Fab Immunoglobulins; Fluorescent Probes; Grant; Health; Human; Human body; Image; Immunofluorescence Immunologic; Industrialization; Injections; Laboratories; Maps; Mass Spectrum Analysis; Measurement; Names; Outcome; Phage Display; Phosphoserine; Phosphotyrosine; Post-Translational Protein Processing; Process; Proteins; Proteolysis; Proteomics; Reagent; Recombinant Antibody; Recombinants; Research Personnel; Resolution; Running; Site; Solid; Source; Spatial Distribution; Specific qualifier value; Specificity; System; Technology; Threonine; Tissues; Ubiquitin; Validation; Work; antibody libraries; cell type; human tissue; imaging modality; interest; microscopic imaging; molecular recognition; overexpression; stoichiometry; Affinity; Antibodies; Automation; Awareness; Bacteriophages; Biological; Cells; Cellular Structures; Chemicals; Colon; Colorectal; Confocal Microscopy; DNA; Data; Development; Epithelial Cells; Escherichia coli; Fab Immunoglobulins; Fluorescent Probes; Grant; Health; Human; Human body; Image; Immunofluorescence Immunologic; Industrialization; Injections; Laboratories; Maps; Mass Spectrum Analysis; Measurement; Names; Outcome; Phage Display; Phosphoserine; Phosphotyrosine; Post-Translational Protein Processing; Process; Proteins; Proteolysis; Proteomics; Reagent; Recombinant Antibody; Recombinants; Research Personnel; Resolution; Running; Site; Solid; Source; Spatial Distribution; Specific qualifier value; Specificity; System; Technology; Threonine; Tissues; Ubiquitin; Validation; Work; antibody libraries; cell type; human tissue; imaging modality; interest; microscopic imaging; molecular recognition; overexpression; stoichiometry

Abstract The mapping of healthy human tissue is an ongoing challenge, critical to which is understanding the cellular distribution and composition of biomolecules. Among these biomolecules, proteins are perhaps the least regularized and deeply understood, with their myriad proteoforms created within cells of distinct types. Here, we outline a new measurement framework poised to better illuminate the underlying cell types and structure defining solid tissues types in human health and wellness. We propose a two-step process that identifies and characterizes selected proteins within cells of human tissue and offers the HuBMAP Consortium highly validated and renewable affinity reagents in the process. Step 1 involves the rapid deployment of previously developed, recombinant affinity reagents (rABs) and the precise characterization of whole proteoforms in human colon tissue recognized by them. Step 2 involves the creation of “smart” probes that work on specific post-translational modifications (PTMs) in a ‘proteoform-aware’ fashion. The result will be specific, renewable and extremely validated affinity probes that are readily disseminated for use by HuBMAP Tissue Mapping Centers (TMCs). Importantly, these recombinant antibodies (rABs) will be among the most characterized of any in terms of their molecular recognition by the end of the three year granting period. This is because of the kind of low-bias readout that employs “top-down” mass spectrometry (TDMS) to interrogate whole proteoforms and their PTMs directly (no proteolysis prior to mass spectrometry). The direct deliverable of this activity will be 30 affinity reagents (20 simple and 10 smart rABs that recognize PTMs) and hundreds of characterized proteoforms to key targets of high interest to HuBMAP. The affinity reagents that emerge will be in a recombinant and renewable form (i.e., proven to express in E. coli) that are ready for use in a variety of imaging modalities through conjugation to fluorescent probes. These will be applicable to immunofluorescence by confocal microscopy, imaging CyTOF (MIBI), and DNA-barcoded antibodies to name a few. The stringent process of development and validation represents a general approach to better map both the chemical space of proteins and their spatial distribution in tissue at cellular and sub- cellular resolution. Our combined platform focuses on the biologically active forms of proteins (proteoforms) and the PTMs decorating them that maintain health and wellness in the human body. PTM maps enabled by smart and renewable reagents represent a high value outcome, templated here through this collaborative effort between the Wells and Kelleher laboratories both coordinating effectively with each other and the HuBMAP Consortium.

抽象的 健康人组织的映射是一个持续的挑战，至关重要的是了解细胞 生物分子的分布和组成。在这些生物分子中，蛋白质可能最少 正规化和深刻理解的，它们在不同类型的细胞中产生的无数蛋白质成型。在这里，我们 概述一个新的测量框架中毒，以更好地阐明基础细胞类型和结构定义 固体组织类型在人类健康和健康中。我们提出了一个两步的过程，可以识别和 表征人类组织细胞中选定的蛋白质，并提供高度验证的Hubmap联盟 以及此过程中可再生的亲和力试剂。步骤1涉及先前开发的快速部署， 重组亲和力试剂（RAB）和人类结肠组织中全蛋白相的精确表征 被他们认可。步骤2涉及在特定翻译后起作用的“智能”问题的创建 修改（PTMS）以“蛋白质成型”的方式进行。结果将是具体的，可再生的，而且非常 经过验证的亲和力问题，这些问题很容易被Hubmap组织映射中心（TMC）使用。 重要的是，这些重组抗体（RAB）将是其最多的特征 分子识别到三年授予期结束。这是因为那种低偏见的读数 员工“自上而下”的质谱（TDM）直接询问整个蛋白质成型型及其PTM （在质谱之前没有蛋白水解）。 这项活动的直接交付将是30种亲和力试剂（20个简单和10个智能Rabs，可以识别 PTMS）和数百种特征性的蛋白质成型，指向Hubmap高度关注的关键靶标。亲和力 出现的试剂将以重组和可再生形式（即证明在大肠杆菌中表达）是 通过结合荧光问题，可以在各种成像方式中使用。这些将是 适用于共聚焦显微镜，成像细胞（MIBI）和DNA-BARCODED的免疫荧光 抗体等。严格的开发和验证过程代表了一种一般方法 为了更好地绘制蛋白质的化学空间及其在细胞和亚蛋白质组织中的空间分布 细胞分辨率。我们的联合平台着重于蛋白质（蛋白质成型）和 PTM装饰它们，以维持人体健康和健康。 smart启用的PTM地图 可再生试剂代表了一个高价值的结果，在这里通过这种协作努力模板 在井和凯勒尔实验室之间，彼此有效地协调 财团。