Laventie Benoît-Joseph, Glatter Timo, Jenal Urs
Infection Biology, Biozentrum, University of Basel, Basel, Switzerland.
Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland.
Methods Mol Biol. 2017;1657:361-376. doi: 10.1007/978-1-4939-7240-1_28.
Capture compound technology coupled to mass spectrometry (CCMS) allows to biochemically identify ligand receptors. Using a c-di-GMP-specific Capture Compound, we adapted this method for the identification and characterization of c-di-GMP binding proteins in any bacterial species. Because in silico analysis often fails to predict novel c-di-GMP effectors, this universal method aims at better defining the cellular c-di-GMP network in a wide range of bacteria. CCMS was successfully applied in several bacterial species (Nesper et al., J Proteom 75:4874-4878, 2012; Steiner et al., EMBO J 32:354-368, 2013; Tschowri et al., Cell 158:1136-1147, 2014; Trampari et al., J Biol Chem 290:24470-24483, 2015; Rotem et al., J Bacteriol 198:127-137, 2015). To outline the detailed protocol and to illustrate its power, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control, as an example. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network.
与质谱联用的捕获化合物技术（CCMS）可对配体受体进行生化鉴定。我们使用一种特异性结合环二鸟苷酸（c-di-GMP）的捕获化合物，将该方法应用于鉴定和表征任何细菌物种中的c-di-GMP结合蛋白。由于计算机分析常常无法预测新的c-di-GMP效应物，这种通用方法旨在更好地定义广泛细菌中的细胞c-di-GMP网络。CCMS已成功应用于多种细菌物种（内斯珀等人，《蛋白质组学杂志》75:4874 - 4878，2012；施泰纳等人，《欧洲分子生物学组织杂志》32:354 - 368，2013；乔维里等人，《细胞》158:1136 - 1147，2014；特兰帕里等人，《生物化学杂志》290:24470 - 24483，2015；罗特姆等人，《细菌学杂志》198:127 - 137，2015）。为了概述详细方案并展示其功效，我们以铜绿假单胞菌为例，它是一种机会致病菌，其中c-di-GMP在毒力和生物膜控制中起关键作用。CCMS鉴定出了c-di-GMP网络中74%（38/51）的已知或预测成分。
