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

Abstract

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 actinbinding 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. Molecular & Cellular Proteomics

Global Analysis Reveals the Complexity of the Human Glomerular Extracellular Matrix

“…Although laser microdissection studies have the ability to precisely define the anatomic structure for analysis, the total number of proteins identified in the most recent of these studies, using a variety of tissue samples, ranged from 114 to 340, which is fewer proteins than the large-scale glomerular studies and significantly fewer ECM proteins compared with this study. 13,34,35 Future developments in technologies, which may couple tissue imaging with improved sensitivity of molecular analysis, may allow direct compositional analysis of ECM within distinct tissue compartments. 36,37 The profile of glomerular ECM that we have described is likely to represent a snapshot of a highly dynamic extracellular environment that changes under the influence of cellular, physical, and chemical environmental cues.…”

Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection

“…The spectral index calculations were subsequently performed as described1718. To reduce false-positive identifications the false discovery rate (FDR) was set on peptide spectrum match (PSM) level to <0.01, the PSM were exported from PIA (https://github.com/mpc-bioinformatics/pia) and further processed using the “pivot table” function in Microsoft Excel, which yielded a table of the spectral counts for every peptide that belonged to a certain protein in a sample.…”

Aciculin interacts with filamin C and Xin and is essential for myofibril assembly, remodeling and maintenance

“…Such repair zones become evident as regions lacking aactinin, titin and nebulin, whereas desmin and actin are enriched (Yu et al, 2004;Yu and Thornell, 2002). They are efficiently revealed by staining for filamin C (FLNc) and its ligands Xin and XIRP2 Kley et al, 2013;Otten et al, 2012;van der Ven et al, 2006). FLNc has also been described as general marker of skeletal muscle damage in numerous neuromuscular diseases (Bönnemann et al, 2003;Sewry et al, 2002;Thompson et al, 2000).…”