Subcellular Microdissection for the Identification of Organelle Proteins

用于鉴定细胞器蛋白的亚细胞显微切割

基本信息

批准号：
7969483
负责人：
SANFORD P MARKEY
金额：
$ 2.34万
依托单位：
NATIONAL INSTITUTE OF MENTAL HEALTH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7969483
关键词：
Address Antibodies Area Award Biological Biological Markers Calcium-Sensing Receptors Categories Cells Chemicals Citrates Complex Computers Coupled Deposition Development Disease Dissection Electron Microscopy Enzymes Fiber Film Functional disorder Genomics Golgi Apparatus Grant Heating Heavy Metals Histologic Horseradish Peroxidase Image In Situ Institutes Label Lasers Lead Location Maps Mass Spectrum Analysis Measurement Metals Methods Microdissection Microscopic Mitochondria Modification Molecular Molecular Analysis Molecular Classification of Tumors Organelles Osmium Osmium Tetroxide Pathology Polymers Process Property Proteins Proteomics Radioactive Reaction Reagent Reliance Resolution Sampling Scanning Scheme Sequence Analysis Site Slice Source Speed Staging Staining method Stains Structure Subcellular structure System Techniques Technology Time Tissue Stains Tissues absorption base ethyl vinyl acetate falls human disease instrument interest melting nanoGold protein complex prototype research study sample fixation ultraviolet

Address Antibodies Area Award Biological Biological Markers Calcium-Sensing Receptors Categories Cells Chemicals Citrates Complex Computers Coupled Deposition Development Disease Dissection Electron Microscopy Enzymes Fiber Film Functional disorder Genomics Golgi Apparatus Grant Heating Heavy Metals Histologic Horseradish Peroxidase Image In Situ Institutes Label Lasers Lead Location Maps Mass Spectrum Analysis Measurement Metals Methods Microdissection Microscopic Mitochondria Modification Molecular Molecular Analysis Molecular Classification of Tumors Organelles Osmium Osmium Tetroxide Pathology Polymers Process Property Proteins Proteomics Radioactive Reaction Reagent Reliance Resolution Sampling Scanning Scheme Sequence Analysis Site Slice Source Speed Staging Staining method Stains Structure Subcellular structure System Techniques Technology Time Tissue Stains Tissues absorption base ethyl vinyl acetate falls human disease instrument interest melting nanoGold protein complex prototype research study sample fixation ultraviolet

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项目摘要

This is a new project, and consequently, proof-of-principle experiments are in progress. Initially, we are exploring expanding the limits of Expression Microdissection (xMD). Existing commercial LCM instruments fall into two broad categories: infrared laser systems which melt an absorbing thermal film in areas illuminated by the laser, capturing nearby tissue; and ultraviolet cutting systems which collect areas circumscribed by the laser. Both use microscopic imaging to define the targets for capture. In contrast, the current prototype system used for xMD requires only a fiber-coupled laser source and a computer-controlled scanning stage. A clear ethyl vinyl acetate (EVA) film is placed over an immunohistologically stained tissue section and a 120 μm laser beam rastered over the entire tissue section. The energy of the laser is absorbed only by the dark stain (DAB), which then results in local melting of the polymer film, thereby capturing only the labeled areas for downstream molecular analysis. The technique is unsupervised, automatically retrieving any antibody-stained region, and relatively rapid: a 2 cm x 3 cm tissue slice can be fully dissected in 30 minutes. In addition, the theoretical resolution is not limited by diffraction but is determined by the melt properties of the film and heat transfer from the absorber. Furthermore, the capture speed and spatial resolution are uncorrelated; dissection of a full tissue slice with 100 nm resolution could theoretically be accomplished in the same time it takes for 100 μm resolution. A significant limitation of the current xMD method for proteomics is its reliance on an immunostaining process that introduces significant chemical contamination for subsequent proteomics. Nevrtheless, we are exploring whether antibody coupled reagents, particularly metal bearing, allow capture and transfer of subcellular organelles. We are also exploring direct use of osmium or lead staining, eliminating the need for a horseradish peroxidase catalyzed reactions. Heavy metals have been widely used in electron microscopy to stain and contrast proteins and organelles. The "black reaction" first described by Camillo Golgi in 1898 results from the reduction of osmium tetroxide to a black precipitate at calcium receptors in the cisternae. Alternatively, lead citrate also stains Golgi due to enzymes ubiquitously present and active after fixation. With either osmium or lead, the spatial resolution will be defined by the extent of the metal deposit, by the contrast in absorption between the stained areas and the background, and by the ability to limit melting within the polymer film. Based on the proteomic analyses with metal staining using classical methods, we will later explore alternative strategies for specific protein metal staining, including immunogold.

这是一个新项目，因此正在进行原理证明实验。最初，我们正在探索扩大表达微分解（XMD）的限制。现有的商业LCM仪器分为两个广泛的类别：红外激光系统，它们在激光照亮的区域融化了吸收的热膜，捕获附近的组织；和紫外线切割系统，收集激光限制的区域。两者都使用微观成像来定义捕获目标。相比之下，用于XMD的当前原型系统仅需要光纤耦合的激光源和计算机控制的扫描阶段。将透明的乙酸乙酯（EVA）膜放置在免疫组织学染色的组织截面上，并在整个组织截面上横冲直撞120μm激光束。激光的能量仅被深色染色（DAB）吸收，然后导致聚合物膜的局部熔化，从而仅捕获标记的区域以进行下游分子分析。该技术是无监督的，会自动检索任何抗体染色区域，并且相对较快：2 cm x 3 cm的组织切片可以在30分钟内完全剖析。另外，理论分辨率不受衍射的限制，而是由膜的熔体特性和吸收剂传热确定。此外，捕获速度和空间分辨率是不相关的。从理论上讲，可以在100μm分辨率的同时完成以100 nm分辨率的全组织切片的解剖。当前XMD方法的蛋白质组学的一个重要局限性是其对免疫染色过程的依赖，该过程引入了对随后的蛋白质组学引入严重的化学污染。但是，我们正在探索抗体偶联试剂，尤其是金属轴承是否允许捕获和转移亚细胞细胞器。我们还正在探索直接使用osmium或铅染色，从而消除了对辣根过氧化物酶催化反应的需求。重金属已在电子显微镜中广泛用于染色和对比蛋白质和细胞器。卡米洛·高尔基（Camillo Golgi）在1898年首先描述的“黑色反应”是由于甲壳虫中钙受体在钙受体下的黑色沉淀物还原为黑色沉淀的结果。另外，柠檬酸铅也因固定后普遍存在和活性而导致的高尔基体染色。使用osmium或铅，空间分辨率将由金属沉积物的程度，染色区域和背景之间的吸收以及限制聚合物膜内熔化的能力来定义。基于使用经典方法进行金属染色的蛋白质组学分析，我们稍后将探讨特定蛋白质金属染色的替代策略，包括免疫金。