Cummins Timothy D, Sapkota Gopal P
Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
Division of Nephrology and Hypertension, Clinical Proteomics Center, University of Louisville School of Medicine, Louisville, KY, USA.
Methods Mol Biol. 2018;1788:43-61. doi: 10.1007/7651_2017_85.
Identification and characterization of large protein complexes is a mainstay of biochemical toolboxes. Utilization of cross-linking chemicals can facilitate the capture and identification of transient or weak interactions of a transient nature (Huang and Kim, PloS One 8:e61430, 2013; Gao et al., J Vis Exp doi: 10.3791/51387, 2014). Here we describe a detailed methodology for a cell culture-based proteomic approach. We describe the generation of cells stably expressing green fluorescent protein (GFP)-tagged proteins under the tetracycline-inducible promoter and subsequent proteomic analysis of GFP-interacting proteins. We include a list of proteins that were identified as interactors of GFP.
大型蛋白质复合物的鉴定与表征是生化工具箱的核心内容。利用交联化学物质有助于捕获和鉴定具有瞬时性质的瞬时或弱相互作用（Huang和Kim，《公共科学图书馆·综合》8：e61430，2013；Gao等人，《可视实验杂志》doi：10.3791/51387，2014）。在此，我们描述一种基于细胞培养的蛋白质组学方法的详细方案。我们描述了在四环素诱导型启动子控制下稳定表达绿色荧光蛋白（GFP）标记蛋白的细胞的生成，以及随后对与GFP相互作用蛋白的蛋白质组学分析。我们列出了被鉴定为GFP相互作用蛋白的蛋白质清单。
