Dataset Information

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

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 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Hek-293 Cell, Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: SHIBU KRISHNAN

LAB HEAD: Professor Giovanni Cuda

PROVIDER: PXD001609 | Pride | 2015-04-07

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	EHZF_18Olabelling_1A-3_150.raw	Raw
	EHZF_18Olabelling_1A-3_500.raw	Raw
	EHZF_18Olabelling_1A-3_75.raw	Raw
	EHZF_18Olabelling_1a-3_250.raw	Raw
	EHZF_18Olabelling_1b-5_150.raw	Raw

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

Bernaudo Francesca F Monteleone Francesca F Mesuraca Maria M Krishnan Shibu S Chiarella Emanuela E Scicchitano Stefania S Cuda Giovanni G Morrone Giovanni G Bond Heather M HM Gaspari Marco M

Journal of proteome research 20150324 4

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 intera ...[more]

PMID: 25774781

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