Renewable and Specific Affinity Reagents for Mapping Proteoforms in Human Tissues

摘要 绘制健康人体组织图是一项持续的挑战，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 规律化和深入理解，它们的无数蛋白质形式在不同类型的细胞中产生。在这里，我们 勾勒出一个新的测量框架，以便更好地阐明基本的单元类型和结构定义 人体健康中的固体组织类型。我们提出了一个分两步进行的过程，即确定和 鉴定人体组织细胞内的选定蛋白质，并为HuBMAP联盟提供高度验证的 以及在此过程中的可再生亲和试剂。步骤1涉及快速部署先前开发的、 重组亲和试剂(RABS)及其在人结肠组织中全蛋白的精确鉴定 得到了他们的认可。步骤2涉及创建工作于特定翻译后处理的“智能”探测器 以蛋白质形式感知的方式进行修饰(PTM)。结果将是具体的、可再生的和极端的 经过验证的亲和探针，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)将是任何重组抗体中最具特征的抗体之一 在三年授权期结束前进行分子识别。这是因为这种低偏置读出 它使用“自上而下”的质谱学(Tdms)来直接询问整个蛋白质形式及其ptm。 (在质谱学之前没有蛋白质分解)。 这项活动的直接交付将是30种亲和试剂(20种简单的和10种识别的智能RAB PTMS)和数百个HuBMAP高度关注的关键靶标的特征蛋白形式。亲和力 出现的试剂将以重组和可再生的形式(即已被证明在大肠杆菌中表达) 通过与荧光探针的结合，可用于各种成像方式。这些将是 适用于共聚焦显微镜、细胞荧光成像(MIBI)和DNA条码的免疫荧光 举几个例子，抗体。严格的开发和验证过程代表了一种通用的方法 为了更好地绘制蛋白质的化学空间及其在细胞和亚细胞组织中的空间分布 细胞分辨率。我们的联合平台专注于具有生物活性的蛋白质形式(蛋白质形式)和 PTM装饰它们，保持人体的健康和健康。SMART支持的PTM地图 可再生试剂代表着高价值的成果，通过这种合作努力在这里建立了模板 Wells和Kelleher实验室之间的有效协调以及HuBMAP 财团。

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.