Uddin Md Jalal, Grunnvåg Jeanette S, Lentz Christian S
Department of Medical Biology and Centre for New Antibacterial Strategies (CANS), UiT The Arctic University of Norway, Tromsø, Norway.
Methods Mol Biol. 2025;2921:57-72. doi: 10.1007/978-1-0716-4502-4_3.
Activity-based protein profiling (ABPP) is a chemoproteomic technique that uses functionalized covalent enzyme inhibitors known as activity-based probes (ABPs) to detect, quantify, and identify active enzymes in a biological sample. Here, we describe experimental protocols for profiling of enzyme activities in bacterial cultures using both fluorescent and biotinylated ABPs that allow for the gel-based detection and resolution of target enzymes, or their enrichment and mass spectrometry-based identification, respectively. These protocols have been optimized for profiling of serine hydrolases using fluorophosphonate probes in both Gram-positive and Gram-negative bacteria, and they can easily be adapted for the application of probes targeting other enzyme families of interest in bacteria under diverse growth conditions.
基于活性的蛋白质谱分析（ABPP）是一种化学蛋白质组学技术，它使用被称为基于活性的探针（ABP）的功能化共价酶抑制剂来检测、定量和鉴定生物样品中的活性酶。在此，我们描述了使用荧光和生物素化ABP对细菌培养物中的酶活性进行谱分析的实验方案，这些方案分别允许基于凝胶的靶酶检测和分离，或其富集及基于质谱的鉴定。这些方案已针对使用氟膦酸酯探针在革兰氏阳性和革兰氏阴性细菌中分析丝氨酸水解酶进行了优化，并且它们可以很容易地适用于在不同生长条件下针对细菌中其他感兴趣的酶家族的探针应用。
