Hereditary inclusion body myopathy (HIBM) is an adult onset, slowly progressive distal and proximal myopathy. Although the causing gene, GNE, encodes for a key enzyme in the biosynthesis of sialic acid, its primary function in HIBM remains unknown. The goal of this study was to unravel new clues on the biological pathways leading to HIBM by proteomic comparison. Muscle cultures and biopsies were analyzed by two dimensional gel electrophoresis (2-DE) and the same biopsy extracts by isobaric tag for relative and absolute quantitation (iTRAQ). Proteins that were differentially expressed in all HIBM specimens versus all controls in each analysis were identified by mass spectrometry. The muscle cultures 2-DE analysis yielded 41 such proteins, while the biopsies 2-DE analysis showed 26 differentially expressed proteins. Out of the 400 proteins identified in biopsies by iTRAQ, 41 showed altered expression. In spite of the different nature of specimens (muscle primary cultures versus muscle biopsies) and of the different methods applied (2D gels versus iTRAQ) the differentially expressed proteins identified in each of the three analyses where related mainly to the same pathways, ubiquitination, stress response and mitochondrial processes, but the most robust cluster (30%) was assigned to cytoskeleton and sarcomere organization. Taken together, these findings indicate a possible novel function of GNE in the muscle filamentous apparatus that could be involved in the pathogenesis of HIBM.