MassIVE MSV000080723

Imported Reanalysis Dataset Public PXD001609

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

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Description

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. The workflow has been optimized concerning: (i) DSP concentration and (ii) incubation times for limited proteolysis, using the stem cell-associated transcription co-factor 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. [dataset license: CC0 1.0 Universal (CC0 1.0)]

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

Contact

Principal Investigators:
(in alphabetical order) 		Professor Giovanni Cuda, University Magna Graecia of Catanzaro, N/A
Submitting User: ccms

Publications

Bernaudo F, Monteleone F, Mesuraca M, Krishnan S, Chiarella E, Scicchitano S, Cuda G, Morrone G, Bond HM, Gaspari M.
Validation of a novel shotgun proteomic workflow for the discovery of protein-protein interactions: focus on ZNF521.
J. Proteome Res. 2015 Apr 3;14(4):1888-99. Epub 2015 Mar 24.

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