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. 2015 Apr 3;14(4):1888-99.
doi: 10.1021/pr501288h. Epub 2015 Mar 24.

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

Francesca Bernaudo  1 , Francesca Monteleone  2 , Maria Mesuraca  1 , Shibu Krishnan  2 , Emanuela Chiarella  1 , Stefania Scicchitano  1 , Giovanni Cuda  2 , Giovanni Morrone  1 , Heather M Bond  1 , Marco Gaspari  2
Affiliations

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

Francesca Bernaudo et al. J Proteome Res. .

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 functional role, such as ZNF423, Spt16, Spt5, were discovered and validated by Western blotting.

Keywords: 18O labeling; AP-MS; NuRD core complex; Spt16; Spt5; ZNF423; ZNF521; interactomics; mass spectrometry; proteomics; shotgun proteomics.

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