2.4. Two-Dimensional Gel Electrophoresis (2DE)

Urinary proteins were separated and analyzed by 2DE. The first-dimension separation was conducted by isoelectrofocusing (IEF) in a PROTEAN IEF^® cell (Bio-Rad Laboratories), mixing 80 μg of proteins from each pool with the lysis buffer (6 M urea, 2 M thiourea, 4% CHAPS, 25 mM DTT, 0.2% ampholytes, all from Bio-Rad Laboratories) to a final volume of 300 μL/sample. The solution was then loaded onto 17-cm immobilized pH gradient (IPG) strips, pH range 3–10 (Ready Strip^TM, Bio-Rad Laboratories), and analyzed as previously reported in detail [18]. The second-dimension separation was performed in a PROTEAN^® II xi cell vertical system (Bio-Rad Laboratories), connected to a refrigerated bath circulator set at constant 10 °C (Cryostatic bath, MPM Instruments S.r.l., MB, Italy). Large size 8–16% polyacrylamide gradient gels (29:1 acrylamide/bis solution, 1.5 M Tris, pH 8.8, 10% SDS, 1% TEMED, 10% ammonium persulfate, from Bio-Rad Laboratories) and TGS 1X running buffer (Bio-Rad Laboratories) were employed for the electrophoretic run. Gels were subsequently incubated overnight at room temperature in a fixing buffer solution (30% ethanol/10% acetic acid, Carlo Erba, Milan, Italy) and then sensitized in the enhancer solution (0.5 M potassium acetate, 0.3% potassium tetrathionate, 30% ethanol, from Merck) before staining with 0.2% silver nitrate (Sigma) for 1 h in the dark. Finally, protein spots were developed by a solution composed of 3% potassium carbonate, 0.03% sodium thiosulfate (Merck), and formaldehyde (Sigma-Aldrich, St. Louis, MI, USA). All reagents and solvents were of analytical grade.

Each gel image was acquired by a calibrated densitometer and exported to the PDQuest 2D image analysis software program, version 7.3.1 (Bio-Rad), to accurately detect the significantly increased or decreased protein spots among the studied groups, based on spot stain intensity and area. The differentially expressed spots were cut from the corresponding gel and stored at −20 °C until their processing for MS analysis.