Human Proteome Dataset Hek293, k652 and HeLa cell lines
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Henrik Zauber
LAB HEAD: Prof. Dr. Matthias Selbach
PROVIDER: PXD002383 | Pride | 2015-07-08
REPOSITORIES: Pride
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Project description:We used quantitative mass spectrometry-based proteomics to unravel global changes in the phosphoproteome upon treatment of ASP14 cells (a Ewing Sarcoma cell line) according to the setup below. The combination of drugs were experimentally shown to infer synergy in terms of cell killing. Three experiments were performed using a triple SILAC setup (Light: Lys0,Arg0; Medium: Lys4,Arg6; and Heavy: Lys8,Arg10): Experiment 1: Light SILAC: Drug combination (90 nM Midostaurin + 20 nM BMS-754807) Medium SILAC: Midostaurin (90 nM) Heavy SILAC: 0.1% DMSO Experiment 2: Light SILAC: Drug combination (90 nM Midostaurin + 20 nM BMS-754807) Medium SILAC: Midostaurin (90 nM) Heavy SILAC: BMS-754807 (20 nM) Experiment 3: Light SILAC: Drug combination (90 nM Midostaurin + 20 nM BMS-754807) Medium SILAC: BMS-754807 (20 nM) Heavy SILAC: 0.1% DMSO ASP14 cells were starved by replacing complete SILAC medium with SILAC minimal medium without serum over night. Cells were treated with drugs as stated above for 2h; then they were stimulated with serum for 20 minutes and harvested thereafter.
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Project description:We applied quantitative mass spectrometry (MS)-based proteomics to study the roles of Cbl and Cbl-b in long-term signaling responses related to neurite outgrowth and differentiation of SH-SY5Y neuroblastoma cells. Using stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tag (TMT)-labeling in combination with off-line high-pH reversed-phase fractionation and LC-MS/MS we analyzed how Cbl and Cbl-b depletion by siRNA affected the proteome, phosphoproteome and ubiquitylome of the neuroblastoma cells. SILAC proteome SILAC (Light Arg0/Lys0, medium Arg6/Lys4, heavy Arg10/Lys8) SH-SY5Y cells were treated with Cbl and Cbl-b or control (GFP) siRNA for 72 hours. For combined stimulation with ligand cocktail (FGF-2, IGF-1 PDGF-BB, TGFα) cells were treated with ligands for 48 h. Samples were analyzed in triplicates with set-up as described below: Set-up 1, 3 replicates (R1-3): Light: siGFP, Heavy: siCbl/siCbl-b Set-up 2, 3 replicates (E1-3): Light: siGFP + ligand cocktail, Medium: siCbl/siCbl-b, Heavy: siCbl/siCbl-b + ligand cocktail TMT phosphoproteome and proteome SH-SY5Y cells were treated with Cbl and Cbl-b siRNA, control (GFP) siRNA or Retinoic acid (RA) for 24 hours. Samples were prepared in triplicates and labelled with TMT10-plex reagents according to the set-up below: TMT10-126: siGFP E1 TMT10-127N: siCbl/siCbl-b E1 TMT10-127C: Retinoic acid E1 TMT10-128N: siGFP E2 TMT10-128C: siCbl/siCbl-b E2 TMT10-129N: Retinoic acid E2 TMT10-129C: siGFP E3 TMT10-130N: siCbl/siCbl-b E3 TMT10-130C: Retinoic acid E3 TMT10-131: Mix of the 9 samples SILAC Ubiquitin pulldown SILAC (Light Arg0/Lys0, heavy Arg10/Lys8) SH-SY5Y cells were treated with Cbl and Cbl-b or control (GFP) siRNA for 24 hours. Samples were analyzed in duplicates with set-up as described below: Light: siGFP, Heavy: siCbl/siCbl-b
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