Proteomic Analysis of Cell/Tumor Lysates

Proteomic analyses were performed as previously described29 (link) in biological replicates (n = 4) and technical duplicates (n = 2). Cell/tumor lysates and secreted sample preparations (10 μg protein) were lysed in SDS sample buffer(2% (w/v), 125 mM Tris-HCl, pH 6.8, 12.5% (v/v) glycerol, 0.02% (w/v) bromphenol blue), electrophoresed by short-range SDS-PAGE (10 × 6 mm), and visualized by Imperial Protein Stain (Invitrogen). Individual samples were excised, destained, reduced, alkylated, and trypsinized as described26 (link). A nanoflow Ultra Performance Liquid Chromatography (UPLC) instrument (Ultimate 3000 RSLCnano, Thermo Fisher Scientific) was coupled on-line to a Linear Trap Quadropole (LTQ) Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) with a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were loaded (Acclaim PepMap100, 5 mm × 300 μm i.d., μ-Precolumn packed with 5 μm C18 beads, Thermo Fisher Scientific) and separated (Acquity UPLC M-Class Peptide BEH130, C18, 1.7 μm, 75 μm × 250 mm, Waters). Data was acquired using Xcalibur software v2.1 (Thermo Fisher Scientific).