Aliquots of muscle homogenate (25 μg per sample) were solubilized in Laemmli sample buffer (BioRad Laboratories, Inc., CA, USA), denatured by boiling at 100°C for 5 min and separated by SDS-PAGE. Molecular weight standards (Precision Plus Protein All Blue Standards, BioRad) were also run on each gel to identify the proteins molecular weights. One-dimensional gels were stained by Coomassie Brilliant Blue R-250 (BioRad), and the stained gel images were semiquantitatively analyzed using the Image Studio Lite 3.1.4 software for Macintosh (LI-COR Biosciences, NE, USA).
Bands of interest were manually excised and sent for identification by peptide mass fingerprint to the Inbiotec S.L. (León, Spain) proteomics laboratory, where the samples were processed and analyzed with a 4800 Proteomics Analyzer matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrometer (ABSciex, MA, USA) according to the previously described methods of Oliván et al [25 ]. A database search on Mascot Generic Files combining MS and MS/MS spectra was performed using Mascot v 2.2 from Matrix Science through the Global Protein Server v 3.6 (ABSciex). When the Mascot score was greater than 85 points, the identified protein was considered a valid candidate.
Bands of interest were manually excised and sent for identification by peptide mass fingerprint to the Inbiotec S.L. (León, Spain) proteomics laboratory, where the samples were processed and analyzed with a 4800 Proteomics Analyzer matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrometer (ABSciex, MA, USA) according to the previously described methods of Oliván et al [25 ]. A database search on Mascot Generic Files combining MS and MS/MS spectra was performed using Mascot v 2.2 from Matrix Science through the Global Protein Server v 3.6 (ABSciex). When the Mascot score was greater than 85 points, the identified protein was considered a valid candidate.