(TIF 1576 kb) Additional file 7:(5.7M, tif) Figure S5.?Immunolocalization of additional chaperones. myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy. Conclusions Our findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0280-0) contains supplementary material, which is available to authorized users. (synonym and mutations. We used our established combined laser microdissection and mass spectrometry approach to identify new disease-relevant proteins that accumulate in abnormal fibers. Extensive immunofluorescence studies were performed to validate our proteomic findings and to get a deeper insight into the subcellular distribution of proteins. Furthermore, we compared the results with our previous findings in filaminopathy and desminopathy in order to identify specific proteomic markers for myotilinopathy. Materials and methods Patients Skeletal muscle samples from 15 myotilinopathy patients with a histological phenotype typical of MFM and with known pathogenic mutations in exon 2 of were included in this study. Two patients carried a p.Lys36Glu mutation [11], one patient a p.Ser55Phe mutation [4, 11], six VU591 patients a p.Ser60Cys mutation [4, VU591 11], and six patients a p.Ser60Phe mutation [4, 11]. More detailed data VU591 of patients and muscle samples are presented in Table?1. Table 1 Overview of myotilinopathy patients included in this study mutationvaluevalue between 0.05 and 0.1 but validated by immunofluorescence studies aper mill of total spectral counts A functional classification of proteins by review of the literature revealed that Z-disc and Z-disc-associated proteins, including the disease causing protein myotilin, were most abundantly over-represented in aggregate samples, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport (Fig.?2). Proteomic profiles of aggregates collected from different muscles were largely homogeneous and independent of the specific mutation (Additional file 3: Figure S1). Open in a separate window Fig. 2 Functional classification and Rabbit Polyclonal to MERTK interactions of proteins identified as over-represented in myotilinopathy aggregates samples. a Results of proteomic analysis in myotilinopathy. Proteins identified as over-represented in aggregate samples by mass spectrometric analysis are grouped with regard to their localization or main function. Shown are proportions in aggregate and control samples. Z-disc (?associated) proteins constituted the most abundant group of over-represented proteins, followed by proteins of the sarcolemma and extracellular matrix (ECM), proteins involved in protein quality control and degradation and proteins with a function in actin dynamics or cytoskeletal transport. b Schematic illustration of over-represented proteins which were verified by immunofluorescence studies and of protein-protein interactions. Each protein is termed by the name of its encoding gene (see Additional file 2: Table S2). Direct protein interactions, identified by review of the literature, are depicted by solid connecting lines. Indirect protein interactions are depicted by a dashed connecting line Comparison of mass spectrometric analysis in different MFM subtypes The comparison of data from proteomic analysis in myotilinopathy, filaminopathy and desminopathy revealed that desmin, filamin C, myotilin, N-RAP, Xin, Xirp2, B-crystallin, and nestin were always over-represented in aggregate samples irrespective of the MFM subtype, although differences in ratios and proportions were detected. The ratio of the myotilin and filamin C proportions in aggregate samples was identified as a new diagnostic marker for myotilinopathy with a high sensitivity (100?%) and specificity (91?%) in our MFM cohort VU591 (Fig.?3). This ratio was always above 0.3 in samples from myotilinopathy patients (range 0.34C1.17) and below 0.3 in all filaminopathy (range 0.07C0.1) and in 4 out of 5 desminopathy patients (range 0.11C0.26). Open in a separate window Fig. 3 Individual myotilin/filamin C ratios in aggregate samples from myotilinopathy, desminopathy and filaminopathy patients. Proportions of myotilin and filamin C in aggregate samples were determined by our proteomic approach. The myotilin/filamin C ratio was always above.