Activity-Based Profiling of Lipases in Living Cells
Activity-Based Profiling of Lipases in Living Cells
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DOI：
10.1007/978-1-60761-325-1_14
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发表时间：
2009-01-01
期刊：
影响因子：
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通讯作者：
Birner-Gruenberger, Ruth
中科院分区：
文献类型：
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作者：
Schicher, Maximilian;Jesse, Iris;Birner-Gruenberger, Ruth
蛋白质组学的最终目标是表征所有蛋白质的功能，并在最生理相关的设置可能。实现这一目标的一个步骤是引入基于活性的蛋白质组学。可以使用基于特异性活性的探针直接在蛋白质组中促进对单个蛋白质活性的同时检测，所述探针由靶向某些酶种类的识别位点、与靶标形成共价键的适当定位的反应位点和用于共价结合的靶标的可视化和/或纯化的报告标签组成。由于报道标签的性质如极性、大小、电荷、结构和化学反应性对探针对靶酶的反应性具有很大影响，因此设计了在酶活性探针结合事件之后适合于报道标签的探针。这些探针更接近天然底物，并且足够小和疏水以穿过活细胞的膜。本文介绍了在完整活细胞中检测脂肪分解活性的方法学，包括探针和报告基因的合成、标记方法和靶酶的检测。
The ultimate goal of proteomics; is to characterize the function of all proteins in parallel and in the most physiologically relevant settings possible. A step toward this goal has been the introduction of activity-based proteomics. The simultaneous detection of individual protein activities can be facilitated directly in the proteome using specific activity-based probes consisting of a recognition site targeting a certain enzyme species, a properly positioned reactive site which forms a covalent bond with the target and a reporter tag for Visualization and/or purification of the covalently bound target. As properties like polarity, size, charge, structure, and chemical reactivity of the reporter tag have a large impact on the reactivity of the probes toward the target enzymes probes suitable for reporter tagging after the enzyme-activity probe-binding event were designed. These probes resemble the natural substrates more closely and are small and hydrophobic enough to cross the membrane of living cells. Here the methodology for detection of lipolytic activities in intact living cells, including synthesis of probe and reporter, labeling procedure, and detection of target enzymes is described.