GFP-DIAPH3 Interactome Analysis by Mass Spectrometry

U87 cells (1.5 × 10⁶), stably expressing GFP or GFP-DIAPH3, were lysed in RIPA buffer and immunoprecipitated with anti-GFP antibodies at 4 ^oC, as above. DU145 cells (1.25 × 10⁶), stably expressing GFP or GFP-DIAPH3, were immuno-precipitated at 4 ^oC or 25 ^oC. Proteins eluted from the beads were separated in a 10% SDS-PAGE gel, and in-gel reduced, alkylated, and tryptically digested56 (link). Tryptic peptides were extracted, concentrated, reconstituted in 0.1% formic acid, separated on a 25 cm EASY-Spray C18 column, and analyzed by an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific). After each survey scan, up to 20 collision-induced dissociation (CID) spectra were acquired in the rapid CID scan mode. For protein identification and quantitation, raw mass spectrometric data were searched against the Uniprot_Human database (released on 02/20/14, including 88647 sequences) with MaxQuant (v 1.3.0.5)57 (link) and Andromeda58 (link). False discovery rates for protein and peptide identifications were set at 0.01. Identified proteins were quantitated based on their summed ion intensities. All data have been deposited into ProteomeXchange59 (link). The DIAPH3 interactome was analyzed using the DAVID bioinformatics database60 (link).