Whole OB Proteome Profiling

Whole OB specimens (70–80 mg) derived from controls and MixD cases were homogenized in lysis buffer containing 7 M urea, 2 M thiourea, 50 mM DTT. After ultracentrifugation, protein extracts were precipitated, pellets were dissolved in 6 M Urea and Tris 100 mM pH 7.8 and Bradford assay kit (Bio-Rad) was used for protein quantitation. Whole proteomes were concentrated in the stacking/resolving SDS-PAGE gel interface. After staining, protein digestion (10 ug) was carried out with trypsin (Promega; 1:20, w/w) at 37 °C for 16 h as previously described [30 (link)]. Prior to LC-MS/MS, peptides were purified and concentrated using C18 Zip Tip Solid Phase Extraction (Millipore, Burlington, MA, USA). Label free LC-MS/MS analyses were performed on an EASY-nLC 1200 liquid chromatography system interfaced with a Q Exactive HF-X mass spectrometer (Thermo Scientific, Waltham, MA, USA). Chromatographic/elution conditions and mass-spectrometry parameters were as previously described [31 (link)]. Data were acquired using Xcalibur software (Thermo Scientific, Waltham, MA, USA).