Proteomic profiling of the mouse diaphragm and refined mass spectrometric analysis of the dystrophic phenotype

Abstract

The diaphragm is a crucial muscle involved in active inspiration and whole body homeostasis. Previous biochemical, immunochemical and cell biological investigations have established the distribution and fibre type-specific expression of key diaphragm proteins. Building on these findings, it was of interest to establish the entire experimentally assessable diaphragm proteome and verify the presence of specific protein isoforms within this specialized subtype of skeletal muscle. A highly sensitive Orbitrap Fusion Tribrid mass spectrometer was used for the systematic identification of the mouse diaphragmassociated protein population. Proteomics established 2925 proteins by high confidence peptide identification. Bioinformatics was used to determine the distribution of the main protein classes, biological processes and subcellular localization within the diaphragm proteome. Following the establishment of the respiratory muscle proteome with special emphasis on protein isoform expression in the contractile apparatus, the extra-sarcomeric cytoskeleton, the extracellular matrix and the excitation-contraction coupling apparatus, the mass spectrometric analysis of the diaphragm was extended to the refined identification of proteome-wide changes in X-linked muscular dystrophy. The comparative mass spectrometric profiling of the dystrophin-deficient diaphragm from the mdx-4cv mouse model of Duchenne muscular dystrophy identified 289 decreased and 468 increased protein species. Bioinformatics was employed to analyse the clustering of changes in protein classes and potential alterations in interaction patterns of proteins involved in metabolism, the contractile apparatus, proteostasis and the extracellular matrix. The detailed pathoproteomic profiling of the mdx-4cv diaphragm suggests highly complex alterations in a variety of crucial cellular processes due to deficiency in the membrane cytoskeletal protein dystrophin.

Emerging proteomic biomarkers of X-linked muscular dystrophy

“…In comparative proteomics, systems bioinformatics plays a crucial role in the establishment of proteome-wide changes in particular protein families, biochemical pathways, cellular signalling cascades and protein interaction networks. The existence of large databanks of already catalogued and characterized skeletal muscle-associated proteins is extremely helpful for more refined proteomic investigations [28][29][30][31]. Routine cell biological, immunochemical and physiological assays are used for the independent verification of proteomic findings and the confirmation of new biomarker candidates.…”

“…Both, focused studies on the mass spectrometric identification and biochemical characterization of the dystrophin-glycoprotein complex, as well as systematic cataloguing studies of muscle tissue and cell lines, have been carried out over the last decade. A summary of major proteomic studies on dystrophin and the dystrophin complexome is provided in Table 1 [28][29][30][34][35][36][37][38][39][40][41][42][43][44][45].…”

“…Building on the initial biochemical characterization of the dystrophin complex by density gradient ultracentrifugation and chemical crosslinking analysis [32,46,47], proteomic studies could confirm the close linkage of dystrophin with the integral glycoprotein β-dystroglycan and its associated extracellular laminin-binding protein α-dystroglycan [34,[38][39][40][41][42][43][44][45]. Cortical actin, syntrophins, dystrobrevins, the α,β,γ,δsarcoglycan complex and laminin subunits are routinely identified by mass spectrometric surveys [28][29][30]. However, the integral protein sarcospan is often only identified by low sequence coverage using peptide mass spectrometry, even in highly enriched subcellular fractions from skeletal muscle [45], which is probably due to its low abundance and highly hydrophobic properties.…”

“…This information could be extremely helpful for the future testing and clinical establishment of novel and more robust biomarkers of Duchenne muscular dystrophy. Table 2 provides an overview of the diversity of protein changes that have been identified by the systematic comparison of various skeletal muscles using highly sensitive mass spectrometric techniques [30,[39][40][41][42]44,45,. As recently reviewed [3], proteomic studies revealed that the dystrophindeficient skeletal musculature undergoes only moderate fibre type shifting on the level of bioenergetic pathways and the contractile apparatus.…”

“…These types of muscle proteins are more appropriately analyzed by one-dimensional gel electrophoretic techniques that can be combined with on-membrane digestion and mass spectrometry [34,43,72,77] or affinity purification and immunoprecipitation approaches [39,41,44,77]. Alternatively, skeletal muscle proteins that are present in crude tissue extracts or subcellular fractions can be conveniently analyzed by bottom-up proteomics, which employs trypsination of total protein complements in combination with liquid chromatography and mass spectrometry [30,45,56,60,62,64,[66][67][68][69]71,[73][74][75][76]78].…”