Spatially-resolved proteomic mapping of living cells

活细胞的空间分辨蛋白质组图谱

• 批准号: 8549487
• 负责人: ALICE Y TING
• 金额: $ 74.75万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-23 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8549487
• 关键词: Apoptosis; Biological Preservation; Biological Process; Biotin; Biotinylation; Caring; Cell physiology; Cells; Cellular biology; Centrifugation; Chemicals; Cytolysis; Diagnosis; Disease; Distant; Drug usage; Endoplasmic Reticulum; Enzymes; Functional disorder; Genetic; Genomics; Harvest; Immunoprecipitation; Label; Lead; Life; Light; Maps; Mass Spectrum Analysis; Medical; Medicine; Membrane; Metabolic; Methods; Microscopy; Mitochondria; Molecular; Molecular Biology; Nature; Neurons; Organelles; Patients; Peroxidases; Phenols; Proteins; Proteome; Proteomics; Radial; Recombinant Proteins; Regulation; Role; Sampling; Site; Streptavidin; Synapses; Synaptic Cleft; Synaptic Transmission; Techniques; Technology; Testing; Therapeutic; Time; Work; base; cellular imaging; density; enzyme substrate; innovation; interest; mitochondrion intermembrane space; neuroligin 1; new technology; novel therapeutics; protein complex; public health relevance; quantum; response; screening; small molecule; spatiotemporal

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.

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，但缺乏细胞中的空间信息。结合这些优势是有变革的 两个字段，生成一个细胞的“图像”，其中定义了每个空间区域的完整分子组成。这样的信息将代表我们对细胞功能的分子理解的量子飞跃，但超出了任何当前技术的范围。该提案描述了一种桥接蛋白质组学和显微镜的变革性新技术，以生成活细胞的第一个空间分辨蛋白质组学图。关键创新是一种非特异性标记酶，我们可以基因靶向活细胞内的任何感兴趣区域。一旦靶向，我们将化学底物添加到细胞中，该细胞被酶转化为短寿命和高反应性分子，该分子将其化学标记其立即的任何蛋白质标记 附近。一旦标记，可以通过常规质谱分离并鉴定标记的蛋白质。因为我们精确地知道在细胞中的位置，非特异性标记酶的靶向（例如突触裂缝），并且因为酶生成的反应性分子具有很小的半径，我们必须检测到的任何化学标记的蛋白质都必须在非特异性Enzeme（E.g.g.g.g. e.g. e.g.）中，并在该突出中。我们建议 使用该技术来绘制许多亚细胞区域的完整蛋白质组成，尤其是在分子水平上熟悉的蛋白质组成，例如突触裂缝，线粒体膜间空间和Organelle-Organelle接触区域。除了推进基本分子和细胞生物学外，该项目还具有许多潜在的医学应用，例如对患者衍生细胞的分析及其对治疗剂的反应。这种分析只能使用当前蛋白质组学研究所需的一小部分细胞材料来阐明疾病和药物的分子机制。就像“基因组医学”现在正在彻底改变医疗服务一样，我们设想该项目将为“蛋白质组学医学”打开大门，该项目将提供有关患者重要生物过程功能/功能障碍的关键新信息。