Characterization of protein complexes using chemical cross-linking coupled electrospray mass spectrometry

TL;DR

This paper presents a detailed methodology combining chemical cross-linking and electrospray mass spectrometry to characterize protein complexes, focusing on capturing transient interactions and identifying protein interactors.

Contribution

It introduces a cell culture-based proteomic approach with GFP-tagging and inducible expression to analyze protein interactions in detail.

Findings

Identified GFP-interacting proteins in cell culture

Demonstrated capture of transient protein interactions

Provided a list of specific protein interactors

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. Here we describe a detailed methodology for 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.