Two pools of plasma samples were obtained by mixing equal proportions of plasma [31 (link)], from the eight diabetic subjects of the EXE group, collected before and after the completion of the training program, and prefractioned using the ProteoMiner™ kit Large-Capacity Kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's protocol. Proteins elution was carried out with urea lysis buffer (8 M urea, 4% CHAPS, 65 mM DTE, and 40 mM Tris base). The protein concentration of each sample was determined according to Bradford [32 (link)]. Two-dimensional electrophoresis (2-DE) was carried out as previously described [33 (link)]. Analytical gels were stained with silver nitrate [34 (link)]. Semipreparative gels for mass spectrometry analysis were stained with Brilliant Blue R-250 (Sigma-Aldrich, Milan, Italy) according to the manufacturer's procedure. Gel images were acquired by the Fluor-S MAX multi-imaging system (Bio-Rad Laboratories Italy, Milan, Italy), and the data were analyzed with the ImageMaster 2D Platinum software. Method for in gel digestion was adapted from Shevchenko et al. [35 (link)] as previously described [36 (link)]. LC-ESI-MS/MS analysis was performed using a Q-TOF microTM mass spectrometer (Micromass, Manchester, UK) as previously described [37 (link)].
Free full text: Click here
