Mitochondrial Proteomic Analysis by 2DE-MS

Mitochondria from 3 × 10⁷ cells were extracted as previously described [33 (link)] and suspended in 8 M urea, 4% CHAPS, 65 mM DTE, and 40 mM Tris base and sonicated for 5 s on ice. Following centrifugation at 21000 g, protein concentration was determined by Bradford assay [38 (link)] and 80 μg of total proteins used for 2-dimensional electrophoresis (2DE). Analytical gels were stained with silver nitrate [39 (link)], while semipreparative gels for mass spectrometry analysis were stained with Brilliant Blue R250 (Sigma-Aldrich, USA). Gel images were acquired by Fluor-S MAX multi-imaging system (Bio-Rad Laboratories Italy, Segrate, Italy), and the data were analysed with ImageMaster 2D Platinum software. Gel digestion was carried out according to Shevchenko's protocol [40 (link)] and LC-ESI-MS/MS analysis was performed using a Q-TOF micro™ mass spectrometer (Micromass, UK) as previously described [41 (link)].