Computational protocol: EWS-FLI-1 creates a cell surface microenvironment conducive to IGF signaling by inducing pappalysin-1

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Protocol publication

[…] The preparation of secreted protein samples, GeLC-MS/MS analysis, and proteomics data analysis were performed essentially as described []. A673 cells were infected with lentiviruses expressing an shRNA against FLI-1 C-terminal region or luciferase (control) and were selected with 2 µg/ml puromycin for 2 days. Cells were washed six times with DMEM without serum. Subsequently, cells were cultured in DMEM without serum for 24 hours and the culture supernatant was harvested. The supernatant was centrifuged, filtered through a 0.45 μm filter (Millipore), and concentrated using a 3,000 Dalton cut-off Amicon Ultra Centrifugal Filter Units (Millipore). The proteins in each sample were fractionated by SDS-PAGE and visualized by Coomassie blue. Each gel lane was divided into six slices, and the proteins in each slice were digested in situ with trypsin (Promega modified) in 40 mM NH4HCO3 overnight at 37°C. The resulting tryptic peptides were analyzed by HPLC-ESI-tandem mass spectrometry (HPLC-ESI-MS/MS) on a Thermo Fisher LTQ Orbitrap Velos mass spectrometer. The Xcalibur raw files were converted to mzXML format using ReAdW and were searched against the UniProtKB/Swiss-Prot human protein database using X! Tandem. The X! Tandem search results were analyzed by the Trans- Proteomic Pipeline [] version 4.3. Peptide/protein identifications were validated by Peptide/ProteinProphet [, ]. […]

Pipeline specifications

Software tools ReAdW, X! Tandem, ProteinProphet
Databases UniProt UniProtKB
Application MS-based untargeted proteomics
Organisms Homo sapiens, Homo sapiens/Mus musculus xenograft
Diseases Bone Neoplasms, Sarcoma, Ewing