Endogenous protein interactomes resolved through immunoprecipitation-coupled quantitative proteomics in cell lines

Abstract

Summary: Immunoprecipitation (IP) of endogenously expressed proteins is one of the most biologically relevant techniques to identify protein-protein interactions. We describe an adaptable IP protocol reliant on a specific antibody to the target protein. We detail a quantitative proteomics workflow for the unbiased identification of co-immunoprecipitating proteins, known collectively as an interactome. This includes protocols for the tryptic digestion, Tandem Mass Tag labeling and fractionation of peptides, and their identification and quantification using liquid chromatography-mass spectrometry including computational and statistical analysis.For complete details on the use and execution of this protocol, please refer to Johnson et al. (2020). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell biology
• Proteomics
• Protein expression and purification
• Mass spectrometry