Validation of a novel shotgun proteomic workflow for the discovery of protein-protein interactions: focus on ZNF521

Journal of Proteome Research
Francesca BernaudoMarco Gaspari

Abstract

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 (18)O 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...Continue Reading

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Citations

Jan 21, 2016·BioMed Research International·Maria MesuracaGiovanni Morrone
Jul 5, 2018·Science Signaling·Jacob G SmithAntonella Riccio
Sep 24, 2020·International Journal of Molecular Sciences·Emanuela ChiarellaMaria Mesuraca
Jun 6, 2018·Frontiers in Endocrinology·Heather M BondGiovanni Morrone
Feb 25, 2021·Cell Reports·Catia AndreassiAntonella Riccio
Aug 28, 2021·International Journal of Molecular Sciences·Emanuela ChiarellaMaria Mesuraca

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