Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP) - a general method for mapping sites of probe modification in proteomes
Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP) - a general method for mapping sites of probe modification in proteomes
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DOI：
10.1038/nprot.2007.194
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Cravatt, Benjamin F.
中科院分区：
文献类型：
--
作者：
Weerapana, Eranthie;Speers, Anna E.;Cravatt, Benjamin F.
基于活性的蛋白质谱分析（ABPP）利用活性定点化学探针直接监测天然生物系统中酶的功能状态。在ABPP实验中，确定酶上探针标记的特定位点仍然是一个主要挑战。在该协议中，我们描述了一个先进的ABPP平台，利用串联正交蛋白水解（TOP）的策略，再加上质谱分析，同时确定探针标记的蛋白质连同它们的确切位点的探针修饰。探针修饰位点的阐明揭示了特定探针蛋白相互作用的分子基础的基本见解。TOP-ABPP方法可应用于任何类型的蛋白质组样品，包括来自体外或体内标记实验的那些，并且与各种化学探针结构相容。完成整个方案，包括关键试剂的化学合成，大约需要8-10天。
Activity-based protein profiling (ABPP) utilizes active site-directed chemical probes to monitor the functional state of enzymes directly in native biological systems. Identification of the specific sites of probe labeling on enzymes remains a major challenge in ABPP experiments. In this protocol, we describe an advanced ABPP platform that utilizes a tandem orthogonal proteolysis (TOP) strategy coupled with mass spectrometric analysis to simultaneously identify probe-labeled proteins together with their exact sites of probe modification. Elucidation of probe modification sites reveals fundamental insights into the molecular basis of specific probe protein interactions. The TOP-ABPP method can be applied to any type of proteomic sample, including those derived from in vitro or in vivo labeling experiments, and is compatible with a variety of chemical probe structures. Completion of the entire protocol, including chemical synthesis of key reagents, requires approximately 8-10 days.