Validation of a novel shotgun proteomic workflow for the discovery of protein-protein

The study of protein-protein interactions is increasingly relying on mass spectrometry (MS). The classical approach of separating immunoprecipitated proteins by SDS-PAGE followed by in-gel digestion is long and labour-intensive. Besides, it is difficult to integrate it with most quantitative MS-based workflows, except for stable isotopic labelling of amino acids in cell culture (SILAC). This work describes a fast, flexible and quantitative workflow for the discovery of novel proteinprotein interactions. A cleavable cross-linker, dithiobis[succinimidyl propionate] (DSP), is utilized to stabilize protein complexes before immunoprecipitation. Protein complex detachment from the antibody is achieved by limited proteolysis. Finally, protein quantitation is performed via 18O labelling.