Wendrich Jos R, Boeren Sjef, Möller Barbara K, Weijers Dolf, De Rybel Bert
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.
Department of Plant Systems Biology, VIB Ghent University, 9052, Ghent, Belgium.
Methods Mol Biol. 2017;1497:147-158. doi: 10.1007/978-1-4939-6469-7_14.
Individual proteins often function as part of a protein complex. The identification of interacting proteins is therefore vital to understand the biological role and function of the studied protein. Here we describe a method for the in vivo identification of nuclear, cytoplasmic, and membrane-associated protein complexes from plant tissues using a strategy of immunoprecipitation followed by tandem mass spectrometry. By performing quantitative mass spectrometry measurements on biological triplicates, relative abundance of proteins in GFP-tagged complexes compared to background controls can be statistically evaluated to identify high-confidence interactors. We detail the entire workflow of this approach.
单个蛋白质通常作为蛋白质复合物的一部分发挥作用。因此，鉴定相互作用的蛋白质对于理解所研究蛋白质的生物学作用和功能至关重要。在这里，我们描述了一种从植物组织中体内鉴定核、细胞质和膜相关蛋白质复合物的方法，该方法采用免疫沉淀后串联质谱的策略。通过对生物学重复样本进行定量质谱测量，可以对绿色荧光蛋白（GFP）标记复合物中蛋白质相对于背景对照的相对丰度进行统计学评估，以鉴定高可信度的相互作用蛋白。我们详细介绍了这种方法的整个工作流程。
