Characterization of Protein Complexes Using Chemical Cross-Linking Coupled Electrospray Mass Spectrometry

Abstract

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.