title: 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 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.
authors.affiliation: †Department of Experimental and Clinical Medicine, Laboratory of Molecular Haematopoiesis and Stem Cell Biology, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Bernaudo
authors.affiliation: ‡Department of Experimental and Clinical Medicine, Proteomics@UMG, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Monteleone
authors.affiliation: †Department of Experimental and Clinical Medicine, Laboratory of Molecular Haematopoiesis and Stem Cell Biology, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Mesuraca
authors.affiliation: ‡Department of Experimental and Clinical Medicine, Proteomics@UMG, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Shibu
authors.initials: S
authors.lastname: Krishnan
authors.affiliation: †Department of Experimental and Clinical Medicine, Laboratory of Molecular Haematopoiesis and Stem Cell Biology, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Chiarella
authors.affiliation: †Department of Experimental and Clinical Medicine, Laboratory of Molecular Haematopoiesis and Stem Cell Biology, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Scicchitano
authors.affiliation: ‡Department of Experimental and Clinical Medicine, Proteomics@UMG, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Cuda
authors.affiliation: †Department of Experimental and Clinical Medicine, Laboratory of Molecular Haematopoiesis and Stem Cell Biology, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Morrone
authors.affiliation: †Department of Experimental and Clinical Medicine, Laboratory of Molecular Haematopoiesis and Stem Cell Biology, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Bond
authors.affiliation: ‡Department of Experimental and Clinical Medicine, Proteomics@UMG, University Magna Graecia, Catanzaro 88100, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Gaspari
mesh_terms: D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010103:Oxygen Isotopes; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods*; D011506:Proteins; D040901:Proteomics / Q000379:methods*; D013388:Succinimides; D014157:Transcription Factors / Q000378:metabolism; D035602:Transcriptional Elongation Factors / Q000378:metabolism; D057188:Workflow
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010103:Oxygen Isotopes; D011506:Proteins; C489489:SUPT16H protein, human; C112224:SUPT5H protein, human; D013388:Succinimides; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C031017:ornithine decarboxylase antizyme; C529598:zinc finger protein 521, human; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex; C011240:dithiobis(succinimidylpropionate)
keywords: 18O labeling; AP-MS; NuRD core complex; Spt16; Spt5; ZNF423; ZNF521; interactomics; mass spectrometry; proteomics; shotgun proteomics