Spatially-resolved proteomic mapping of living cells

描述（由申请人提供）：显微镜和蛋白质组学都彻底改变了我们对细胞生物学的理解：虽然显微镜一次为少量蛋白质提供精确的空间信息，但蛋白质组学方法可以检测数千种蛋白质，但缺乏细胞内的空间信息。将这些优势联合收割机结合起来， 两个场，以产生细胞的“图像”，其中每个空间区域的完整分子组成被定义。这些信息将代表我们对细胞功能的分子理解的一个飞跃，但超出了任何现有技术的范围。该提案描述了一种变革性的新技术，用于连接蛋白质组学和显微镜，以产生第一个活细胞的空间分辨蛋白质组图谱。关键的创新是一种非特异性标记酶，我们可以在基因上靶向活细胞内的任何感兴趣区域。一旦有了目标，我们就向细胞中加入一种化学底物，这种底物被酶转化为一种寿命短、反应性强的分子，这种分子可以化学标记其直接作用的任何蛋白质。 附近。一旦标记，标记的蛋白质可以通过常规质谱法分离和鉴定。因为我们精确地知道非特异性标记酶在细胞中的靶向位置（例如突触间隙），并且因为酶产生的反应分子具有非常小的标记半径，所以我们检测到的任何化学标记的蛋白质必须位于非特异性酶附近（例如，在这个例子中的突触间隙中）。我们建议 利用这项技术绘制许多亚细胞区域的完整蛋白质组成图，特别关注那些在分子水平上知之甚少的区域，如突触间隙、线粒体膜间隙和细胞器-细胞器接触区。除了推进基础分子和细胞生物学，该项目还具有许多潜在的医学应用，例如分析患者来源的细胞及其对治疗的反应。这种分析可以揭示疾病和药物的分子机制，只使用目前蛋白质组学研究所需的一小部分细胞材料。正如“基因组医学”现在正在彻底改变医疗保健，我们设想，这个项目将打开大门，“蛋白质组医学”，将提供一个关键的新层的信息有关的功能/功能障碍的重要生物过程中的病人。

DESCRIPTION (provided by applicant): Microscopy and proteomics have both revolutionized our understanding of cell biology: while microscopy provides precise spatial information for small numbers of proteins at a time, proteomic methods can detect thousands of proteins but lack spatial information within cells. It would be transformative to combine the strengths of these two fields, to generate an "image" of the cell in which the complete molecular composition of every spatial region is defined. Such information would represent a quantum leap in our molecular understanding of cellular function, but is beyond the reach of any current technology. This proposal describes a transformative new technology to bridge proteomics and microscopy, to produce the first spatially-resolved proteomic maps of living cells. The key innovation is a nonspecific labeling enzyme that we can genetically target to any region of interest within live cells. Once targeted, we add a chemical substrate to the cell that is converted by the enzyme into a short-lived and highly reactive molecule that chemically labels any protein in its immediate vicinity. Once tagged, the labeled proteins can be isolated and identified by conventional mass spectrometry. Because we know precisely where in the cell the nonspecific labeling enzyme was targeted (e.g. the synaptic cleft), and because the enzyme-generated reactive molecule has a very small labeling radius, any chemically labeled protein that we detect must reside in the vicinity of the nonspecific enzyme (e.g., in the synaptic cleft in this example). We propose to use this technology to map the complete protein composition of many subcellular regions, focusing particularly on those which are poorly understood at the molecular level - such as the synaptic cleft, the mitochondrial inter-membrane space, and organelle-organelle contact zones. In addition to advancing basic molecular and cell biology, this project has many potential medical applications, such as analysis of patient-derived cells and their responses to therapeutics. Such analysis could shed light on the molecular mechanisms of both disease and drug, using only a small fraction of the cellular material required for current proteomic studies. Just as "genomic medicine" is now revolutionizing medical care, we envision that this project will open the door to "proteomic medicine" that will provide a critical new layer of information regarding the function/dysfunction of important biological processes in patients.