Validation of a Novel Shotgun Proteomic Workflow for the Discovery of Protein–Protein Interactions: Focus on ZNF521

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 labor-intensive. Besides, it is difficult to integrate
it with most quantitative MS-based workflows, except for stable isotopic
labeling of amino acids in cell culture (SILAC). This work describes
a fast, flexible and quantitative workflow for the discovery of novel
protein–protein 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 labeling. The workflow has been optimized
concerning (i) DSP concentration and (ii) incubation times for limited
proteolysis, using the stem cell-associated transcription cofactor
ZNF521 as a model target. The interaction of ZNF521 with the core
components of the nuclear remodelling and histone deacetylase (NuRD)
complex, already reported in the literature, was confirmed. Additionally,
interactions with newly discovered molecular partners of potentially
relevant functional role, such as ZNF423, Spt16, Spt5, were discovered
and validated by Western blotting.