Schicher Maximilian, Jesse Iris, Birner-Gruenberger Ruth
Institute of Biochemistry, University of Graz, Graz, Austria.
Methods Mol Biol. 2009;580:251-66. doi: 10.1007/978-1-60761-325-1_14.
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.
蛋白质组学的最终目标是在尽可能与生理相关的环境中并行表征所有蛋白质的功能。朝着这个目标迈出的一步是引入了基于活性的蛋白质组学。使用由靶向特定酶种类的识别位点、与靶标形成共价键的适当定位的反应位点以及用于可视化和/或纯化共价结合靶标的报告标签组成的特定基于活性的探针，可以直接在蛋白质组中促进对单个蛋白质活性的同时检测。由于报告标签的极性、大小、电荷、结构和化学反应性等性质对探针与靶标酶的反应性有很大影响，因此设计了适合在酶活性探针结合事件后进行报告标签标记的探针。这些探针更接近天然底物，并且足够小且疏水以穿过活细胞的膜。本文描述了在完整活细胞中检测脂解活性的方法，包括探针和报告分子的合成、标记程序以及靶标酶的检测。
