Kaschani Farnusch, Gu Christian, van der Hoorn Renier A L
The Plant Chemetics Laboratory, Chemical Genomics Centre, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
Methods Mol Biol. 2012;835:47-59. doi: 10.1007/978-1-61779-501-5_3.
Activity-based protein profiling (ABPP) is a powerful analytical method to detect and compare the activity of proteins in proteomes. This is achieved using specific activity-based probes that are often derived from inhibitors and are linked to reporter groups like rhodamine or biotin for fluorescence detection and/or affinity purification, respectively. The probes react with the active site residue of proteins and become covalently and irreversibly attached, facilitating the separation, detection and identification of the labelled proteins. In this protocol we describe all the steps required for labelling, purification and identification of labelled proteins from gels and show how activities in two proteomes can be compared. The identification of serine hydrolases from Arabidopsis plants infected with Botrytis cinerea using the trifunctional probe TriFP is used as an example.
基于活性的蛋白质谱分析（ABPP）是一种用于检测和比较蛋白质组中蛋白质活性的强大分析方法。这是通过使用特定的基于活性的探针来实现的，这些探针通常衍生自抑制剂，并分别与罗丹明或生物素等报告基团相连，用于荧光检测和/或亲和纯化。探针与蛋白质的活性位点残基发生反应，并共价且不可逆地结合，从而便于对标记蛋白质进行分离、检测和鉴定。在本实验方案中，我们描述了从凝胶中标记、纯化和鉴定标记蛋白质所需的所有步骤，并展示了如何比较两个蛋白质组中的活性。以使用三功能探针TriFP从感染灰葡萄孢的拟南芥植物中鉴定丝氨酸水解酶为例。
