Myofibrillar myopathies (MFM) are histopathologically characterized by desmin-positive protein aggregates and myofibrillar degeneration. While about half of all MFM are caused by mutations in genes encoding sarcomeric and extra-sarcomeric proteins (desmin, filamin C, plectin, VCP, FHL1, ZASP, myotilin, αB-crystallin, and BAG3), the other half of these diseases is due to still unresolved gene defects. The present study aims at the proteomic characterization of pathological protein aggregates in skeletal muscle biopsies from patients with MFM-causing gene mutations. The technical strategy is based on the dissection of plaque- vs. plaque-free tissue areas from the same individual patient by laser dissection microscopy, filter-aided sample preparation, iTRAQ-labeling and analysis on the peptide level using offline nano-LC and MALDI-TOF-TOF tandem mass spectrometry for protein identification and quantification. The outlined workflow overcomes limitations of merely qualitative analyses, which cannot discriminate contaminating non-aggregated proteins. Dependent on the MFM causing mutation different sets of proteins were revealed as genuine (accumulated) plaque components in independent technical replicates: (1) αB-crystallin, desmin, filamin A/C, myotilin, PRAF3, RTN1, SQSTM, XIRP1 and XIRP2 (patient with defined MFM mutation distinct from FHL1) or (2) desmin, FHL1, filamin A/C, KBTBD10, NRAP, SQSTM, RL40, XIRP1 and XIRP2 (patient with FHL1 mutation). The results from differential proteomics indicate that plaques from different patients exhibit protein compositions with partial overlap, on the one hand, and mutation-dependent protein contents on the other. The FHL1 mutation-specific pattern was validated for four patients with respect to desmin, SQSTM, and FHL1 by immunohisto- chemistry.