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Validation of a novel shotgun proteomic workflow for the discovery of protein-protein interactions: focus on ZNF521
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.
Sample Processing Protocol
This present work reports an original workflow for the discovery of novel protein-protein interactions based on: (i) reversible DSP-based cross-linking to stabilize labile interactions and to maximise the recovery of the whole protein complex; (ii) release of the protein complex by onbeads digestion, which allows subsequent gel-free, quantitative mass spectrometric analysis; (iii) 18O labelling to allow simultaneous analysis of IP and corresponding experimental controls and to discriminate between specific interactors and contaminants. DSP concentration and conditions for on-beads digestion have been optimized using the stem cell-associated protein ZNF521, a 30- zinc finger protein that is believed to represent an important cell fate determinant in diverse types of progenitor cells, as a model target. The workflow has been subsequently applied to the analysis of the ZNF521 interactome in HEK293T cells.
Data Processing Protocol
Data were processed using Proteome Discoverer 1.3 (Thermo Fisher Scientific, Bremen, Germany), using Sequest as search engine, and the HUMAN Uniprot protein sequence database (http://www.ebi.ac.uk/uniprot) accessed on March 2013 as sequence database (with isoforms, 87489 entries). The following search parameters were used: MS tolerance 15 ppm; MS/MS tolerance 0.02 Da; fixed modifications carbamidomethyl cysteine; enzyme trypsin; max. missed cleavages 1; variable modifications: oxidised methionine, C-terminal 18O labelling. Peptide identifications were validated by Percolator,36 integrated in Proteome Discoverer. Percolator qvalue was set to equal or less than 0.05. Nevertheless, only proteins identified with at least one peptide having a q-value equal or less than 0.01 were considered for further analysis (but for quantification, peptides with a q-value between 0.01 and 0.05 were also utilized). For label-free experiments (optimization of DSP concentration and optimization of on-beads digestion), quantification at the peptide level was achieved within Proteome Discoverer by using the event detector (mass precision 2 ppm) and precursor ion area detector modules to calculate peptide peak areas. The sum of all peptide peak areas assigned to ZNF521 or any other protein was used as a semi-quantitative measure of protein abundance. For the final experiment (discovery of novel ZNF521 interactors), based on 18O labelling, quantification was achieved within Proteome Discoverer by using the event detector (mass precision 2 ppm) and precursor ion area quantifier (full 18O labelling) modules to calculate peptide “heavy:light” (H:L) ratios. Protein H:L ratios were calculated by taking the median value of all peptide H:L ratios assigned to a specific protein hit.
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 PubMed: 25774781
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||42405||ZNF521ΔNBD Vs Control||2732||18228||2156||92906||5164||
|2||42404||ZNF521ΔNBD Vs Control||2748||18264||2083||89781||4984||
|3||42403||ZNF521 full length Vs Control||2428||19003||2078||93591||5370||
|4||42402||ZNF521 full length Vs Control, Set 2||3152||20238||2387||94770||5578||