Spatially-resolved proteomic mapping of living cells

活细胞的空间分辨蛋白质组图谱

• 批准号: 8739640
• 负责人: ALICE Y TING
• 金额: $ 60.18万
• 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/8739640
• 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：虽然显微镜一次只能提供少量蛋白质的精确空间信息，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。将这些优势结合起来将是一场变革