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

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.

摘要 健康人体组织的映射是一个持续的挑战，其中关键是了解细胞 生物分子的分布和组成。在这些生物分子中，蛋白质可能是最少的 它们在不同类型的细胞内产生了无数的蛋白质形式，这些蛋白质形式被规则化并被深入理解。这里我们 概述了一个新的测量框架，准备更好地阐明潜在的细胞类型和结构定义 固体组织类型在人类健康和福祉。我们提出了一个两步的过程，确定和 表征人体组织细胞内的选定蛋白质，并为HuBMAP联盟提供高度验证 和可再生的亲和试剂。第1步是快速部署以前制定的， 重组亲和试剂（rAB）和人结肠组织中完整蛋白质型的精确表征 被他们认出来。第二步是创建“智能”探针，用于特定的翻译后 修饰（PTM）以“蛋白质形式感知”的方式。其结果将是具体的，可再生的， 经验证的亲和探针，易于传播供HuBMAP组织图谱中心（TMC）使用。 重要的是，这些重组抗体（rAB）将是就其生物学特性而言最具特征的抗体之一。 在三年的授予期结束时获得分子识别。这是因为这种低偏置读出 采用“自上而下”质谱法（TDMS）直接询问整个蛋白质组及其PTM (no在质谱法之前进行蛋白水解）。 这项活动的直接交付将是30种亲和试剂（20种简单和10种智能rAB， PTM）和数百种表征的蛋白质型，用于HuBMAP高度关注的关键靶标。亲和 出现的试剂将是重组的和可再生的形式（即，在E.大肠杆菌）， 通过与荧光探针偶联可用于多种成像方式。这些将是 适用于通过共聚焦显微镜、CyTOF成像（MIBI）和DNA条形码 抗体，仅举几例。严格的开发和验证过程代表了一种通用方法 为了更好地绘制蛋白质的化学空间及其在细胞和亚细胞的组织中的空间分布， 细胞分辨率我们的组合平台专注于蛋白质的生物活性形式（蛋白质形式）， 装饰它们的PTM保持人体的健康和健康。智能支持的PTM映射 和可再生试剂代表了高价值的成果，通过这种合作努力， 在威尔斯和凯莱赫实验室之间进行有效的相互协调， 财团