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.

抽象的 绘制健康人体组织图谱是一项持续的挑战，其中的关键是了解细胞 生物分子的分布和组成。在这些生物分子中，蛋白质可能是最不重要的。 它们在不同类型的细胞内产生了无数的蛋白质形式，从而被规则化并被深入理解。在这里，我们 概述了一个新的测量框架，旨在更好地阐明底层细胞类型和结构定义 人类健康和保健中的固体组织类型。我们提出了一个两步流程来识别和 表征人体组织细胞内选定的蛋白质，并为 HuBMAP Consortium 提供高度验证 以及过程中的可再生亲和试剂。第 1 步涉及快速部署先前开发的、 重组亲和试剂 (rAB) 和人类结肠组织中完整蛋白型的精确表征 得到他们的认可。第 2 步涉及创建作用于特定翻译后蛋白的“智能”探针 以“蛋白质形式感知”的方式进行修饰（PTM）。结果将是具体的、可再生的和极其 经过验证的亲和力探针很容易传播供 HuBMAP 组织作图中心 (TMC) 使用。 重要的是，这些重组抗体 (rAB) 将是最具特征性的抗体之一 在三年授予期结束时进行分子识别。这是因为低偏置读数 采用“自上而下”质谱 (TDMS) 直接询问整个蛋白质组及其 PTM （质谱分析前无蛋白水解）。 该活动的直接成果将是 30 种亲和试剂（20 种简单 rAB 和 10 种智能 rAB，可识别 PTM）和数百种特征化的蛋白质组，针对 HuBMAP 高度感兴趣的关键靶点。亲和力 出现的试剂将采用重组和可再生的形式（即，证明可以在大肠杆菌中表达） 通过与荧光探针结合，可用于各种成像模式。这些将是 适用于共聚焦显微镜免疫荧光、CyTOF (MIBI) 成像和 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 更好地绘制蛋白质的化学空间及其在细胞和亚组织中的空间分布 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）和 装饰它们的 PTM 可以维持人体的健康。智能启用的 PTM 地图 可再生试剂代表了高价值的成果，通过这种协作努力在此进行了模板化 Wells 和 Kelleher 实验室之间有效地相互协调以及 HuBMAP 财团。