A. Maerkens

A Combined Laser Microdissection and Mass Spectrometry Approach Reveals New Disease Relevant Proteins Accumulating in Aggregates of Filaminopathy Patients

Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actin-binding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation.

Differential proteomic analysis of abnormal intramyoplasmic aggregates in desminopathy.

UNLABELLED Desminopathy is a subtype of myofibrillar myopathy caused by desmin mutations and characterized by protein aggregates accumulating in muscle fibers. The aim of this study was to assess the protein composition of these aggregates. Aggregates and intact myofiber sections were obtained from skeletal muscle biopsies of five desminopathy patients by laser microdissection and analyzed by a label-free spectral count-based proteomic approach. We identified 397 proteins with 22 showing significantly higher spectral indices in aggregates (ratio >1.8, p<0.05). Fifteen of these proteins not previously reported as specific aggregate components provide new insights regarding pathomechanisms of desminopathy. Results of proteomic analysis were supported by immunolocalization studies and parallel reaction monitoring. Three mutant desmin variants were detected directly on the protein level as components of the aggregates, suggesting their direct involvement in aggregate-formation and demonstrating for the first time that proteomic analysis can be used for direct identification of a disease-causing mutation in myofibrillar myopathy. Comparison of the proteomic results in desminopathy with our previous analysis of aggregate composition in filaminopathy, another myofibrillar myopathy subtype, allows to determine subtype-specific proteomic profile that facilitates identification of the specific disorder. BIOLOGICAL SIGNIFICANCE Our proteomic analysis provides essential new insights in the composition of pathological protein aggregates in skeletal muscle fibers of desminopathy patients. The results contribute to a better understanding of pathomechanisms in myofibrillar myopathies and provide the basis for hypothesis-driven studies. The detection of specific proteomic profiles in different myofibrillar myopathy subtypes indicates that proteomic analysis may become a useful tool in differential diagnosis of protein aggregate myopathies.

PIA: An Intuitive Protein Inference Engine with a Web-Based User Interface.

Protein inference connects the peptide spectrum matches (PSMs) obtained from database search engines back to proteins, which are typically at the heart of most proteomics studies. Different search engines yield different PSMs and thus different protein lists. Analysis of results from one or multiple search engines is often hampered by different data exchange formats and lack of convenient and intuitive user interfaces. We present PIA, a flexible software suite for combining PSMs from different search engine runs and turning these into consistent results. PIA can be integrated into proteomics data analysis workflows in several ways. A user-friendly graphical user interface can be run either locally or (e.g., for larger core facilities) from a central server. For automated data processing, stand-alone tools are available. PIA implements several established protein inference algorithms and can combine results from different search engines seamlessly. On several benchmark data sets, we show that PIA can identify a larger number of proteins at the same protein FDR when compared to that using inference based on a single search engine. PIA supports the majority of established search engines and data in the mzIdentML standard format. It is implemented in Java and freely available at https://github.com/mpc-bioinformatics/pia.

Myofibrillar instability exacerbated by acute exercise in filaminopathy

Filamin C (FLNC) mutations in humans cause myofibrillar myopathy (MFM) and cardiomyopathy, characterized by protein aggregation and myofibrillar degeneration. We generated the first patient-mimicking knock-in mouse harbouring the most common disease-causing filamin C mutation (p.W2710X). These heterozygous mice developed muscle weakness and myofibrillar instability, with formation of filamin C- and Xin-positive lesions streaming between Z-discs. These lesions, which are distinct from the classical MFM protein aggregates by their morphology and filamentous appearance, were greatly increased in number upon acute physical exercise in the mice. This pathology suggests that mutant filamin influences the mechanical stability of myofibrillar Z-discs, explaining the muscle weakness in mice and humans. Re-evaluation of biopsies from MFM-filaminopathy patients with different FLNC mutations revealed a similar, previously unreported lesion pathology, in addition to the classical protein aggregates, and suggested that structures previously interpreted as aggregates may be in part sarcomeric lesions. We postulate that these lesions define preclinical disease stages, preceding the formation of protein aggregates.

Proteomics of rimmed vacuoles define new risk allele in inclusion body myositis

Sporadic inclusion body myositis (sIBM) pathogenesis is unknown; however, rimmed vacuoles (RVs) are a constant feature. We propose to identify proteins that accumulate within RVs.

A.P.2

Hereditary myopathy with early respiratory failure (HMERF) is caused by A-band titin mutations and characterized by myofibrillar alterations and protein aggregation in skeletal muscle fibers. Our previous studies revealed that the composition of myotilin-positive aggregates is comparable to that seen in myofibrillar myopathies (MFM). However, there are two different types of protein aggregates in HMERF: subsarcolemmal cytoplasmic bodies (CB) with a dense filamentous core and non-CB aggregates. The aim of this study was to compare the proteomic profile of these protein deposits. We used Gomori trichrome stained skeletal muscle sections from two HMERF patients to separately collect CB, non-CB aggregates and intraindividual control samples by laser microdissection and performed a highly sensitive label-free mass spectrometry approach for protein detection and relative quantification based on spectral counting. The proteomic profiles of CB and non-CB aggregates showed a high degree of similarity: XIRP2, desmin, filamin C, N-RAP, nestin, COL6A3, myotilin, sarcosin, αB-crystallin, dystrophin, XIN and aciculin ranked among the most abundant over-represented proteins (ratio >1.5 compared to control samples) in both aggregate types. Peptides assigned to these twelve proteins accounted for 69% (CB) and 75% (non-CB aggregates) of all peptides assigned to over-represented proteins and the individual proportions of these proteins were almost the same. We also detected similarities in less abundant over-represented proteins. Titin was under-represented in CB (ratio 0.81) and in non-CB aggregates (ratio 0.80). In conclusion, our analysis revealed that the proteomic profiles of CB and non-CB aggregates are highly similar in HMERF, especially regarding abundant over-represented proteins, and typical of MFM aggregates.

P.15.6 Desminopathy or myotilinopathy? An integrated proteomics approach for diagnosis

Protein aggregation in skeletal muscle fibers is a hallmark of myofibrillar myopathies and relevant in pathogenesis. Our previous proteomic studies deciphered details of aggregate composition and revealed specific proteomic profiles in different MFM subtypes. The aim of this study was to validate if these profiles are helpful in differential diagnosis in a patient with new mutations in two different MFM genes. The index patient presented with adult-onset weakness affecting limb and respiratory muscles. Two novel and heterozygous nucleotide exchanges in exon 1 of DES and exon 2 of MYOT, both predicted missense mutations, were identified and MFM was proven on the muscle biopsy. Protein aggregates from abnormal fibers and control samples from normally looking muscle fibers were collected by laser microdissection and analyzed by a combination of mass spectrometry (LC-MS/MS) and spectral index calculation. Our proteomic approach detected 113 proteins that were over-represented in intramyoplasmic aggregates of the index patient with a ratio >1.8 compared to control sample. The proteomic profile was consistent with desminopathy: desmin, filamin C, XIRP2, N-Rap and α B-crystallin showed the highest spectral indices of over-represented proteins. Accumulation of the desmin binding partner desmuslin also pointed to a pathogenic desmin mutation. The myotilinopathy markers plectin and obscurin were not over-represented in aggregates of the index patient and spectral index and ratio of myotilin also argued against a MFM-causing MYOT mutation. The results of our study indicate that the index patient harbors a disease-causing desmin mutation and demonstrate that our combined laser microdissection and mass spectrometric approach is a helpful new tool in differential diagnostics of MFM patients. Subtype-specific proteomic profiles can contribute to evaluate the pathogenicity of new mutations in MFM genes.

G.O.5 Deciphering protein aggregates in myofibrillar myopathies – A proteomics approach

Abstract Myofibrillar myopathies (MFM) encompass a genetic heterogenous group of muscle disorders characterized by formation of intracellular protein aggregates in skeletal muscle fibers. We applied a proteomic approach to decipher the aggregate composition in MFM subtypes with the aim to identify novel disease-relevant proteins, disease-specific proteomic profiles and new candidates for MFM-causing proteins. Muscle biopsies of 21 genetically clarified MFM patients (filaminopathy, myotilinopathy, desminopathy, ZASPopathy) were analyzed. Aggregates and intraindividual controls (muscle fibers without aggregates) were collected by laser microdissection. A label-free mass spectrometric approach was used for identification and relative quantification of proteins. A total of 588 different proteins were identified. Up to 193 proteins showed an accumulation in protein aggregates in the different MFM subtypes (ratio >1.8 compared to intraindividual controls). Many of these proteins have never been described in the context of MFM so far. A subset of proteins including desmin, filamin C, Xin, Xirp2 and many other proteins was detected in aggregates in all patients. The comparison of MFM subtypes revealed disease-specific patterns of aggregate compositions with clear differences in ratios of most abundant proteins. Filamin C, desmin, myotilin and ZASP showed the highest accumulation in aggregates of related MFM subtypes. Our proteomic data provide essential new insights in the composition of pathological protein aggregates in MFM. Proteomic profiles of aggregates seem to be specific for the different MFM subtypes and expand our knowledge about proteins involved in pathogenesis of filaminopathy, desminopathy, myotilinopathy and ZASPopathy. The list of abundant proteins in aggregates also include potential new MFM proteins. These should to be considered in future genetic studies in MFM patients with so far unknown mutation.

G.P.57 Differential proteomic analysis of protein aggregates in desminopathy

Abstract Desminopathy is a subtype of myofibrillar myopathies (MFM) caused by mutations in DES, the gene encoding desmin. A histopathologic hallmark of the disease is a massive protein aggregation within skeletal muscle fibers. The aim of our study was to elucidate the composition of aggregates in MFM patients with different desmin mutations by using a label-free mass spectrometry approach. Aggregates and control tissue from muscle biopsies of MFM patients with four different mutations in DES were collected by laser microdissection and analyzed by a combination of mass-spectrometry and spectral index calculation. Proteins with a ratio >1.8 (sum of peptides identified in aggregates compared to intraindividual controls) were accepted as accumulated in aggregates. Results of selected proteins were validated by immunofluorescence studies. Mass spectrometric data were searched against an extended human protein database to detect desmin mutations at the protein level. Three hundred and seventeen different proteins were identified and 98 of them showed an accumulation in aggregates. Aggregate compositions were more heterogenous than in other MFM subtypes, depending on individual mutations, but desmin was on top of the list of abundant proteins in all cases except of one. Immunolocalization findings were consistent with proteomic data. Three out of four desmin mutations were identified at the protein level. Our proteomic approach enabled the identification of many novel components of pathologic protein aggregates within skeletal muscle fibers of desminopathy patients. This provides new insights in the pathogenesis of the disease. Differences in aggregate composition in patients with different desmin mutations indicate diverse pathomechanisms, consistent with data of previous functional studies.