Molecular Fingerprint of BMD Patients Lacking a Portion in the Rod Domain of Dystrophin

Abstract

BMD is characterized by a marked heterogeneity of gene mutations resulting in many abnormal dystrophin proteins with different expression and residual functions. The smaller dystrophin molecules lacking a portion around exon 48 of the rod domain, named the D8 region, are related to milder phenotypes. The study aimed to determine which proteins might contribute to preserving muscle function in these patients. Patients were subdivided, based on the absence or presence of deletions in the D8 region, into two groups, BMD1 and BMD2. Muscle extracts were analyzed by 2-D DIGE, label-free LC-ESI-MS/MS, and Ingenuity pathway analysis (IPA). Increased levels of proteins typical of fast fibers and of proteins involved in the sarcomere reorganization characterize BMD2. IPA of proteomics datasets indicated in BMD2 prevalence of glycolysis and gluconeogenesis and a correct flux through the TCA cycle enabling them to maintain both metabolism and epithelial adherens junction. A 2-D DIGE analysis revealed an increase of acetylated proteoforms of moonlighting proteins aldolase, enolase, and glyceraldehyde-3-phosphate dehydrogenase that can target the nucleus promoting stem cell recruitment and muscle regeneration. In BMD2, immunoblotting indicated higher levels of myogenin and lower levels of PAX7 and SIRT1/2 associated with a set of proteins identified by proteomics as involved in muscle homeostasis maintenance.

Space Omics and Tissue Response in Astronaut Skeletal Muscle after Short and Long Duration Missions

“…Protein extracts (200 µg for each sample) were processed following the filter-aided sample preparation (FASP) protocol [ 66 ]. Peptide samples were concentrated, and separated on a Dionex UltiMate 3000 HPLC System with an Easy Spray PepMap RSLC C18 column (250 mm, internal diameter of 75 µm) (Thermo Fisher Scientific, Rodano, Italy), and electrosprayed into an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, Rodano, Italy) mass spectrometer, as previously described [ 67 ]. Three technical replicates for each sample were acquired.…”

Proteomic profiling of impaired excitation–contraction coupling and abnormal calcium handling in muscular dystrophy

“…Based on the knowledge generated by proteomic cataloguing studies, a considerable number of comparative and mass spectrometry‐based investigations have determined proteome‐wide changes in various types of dystrophic skeletal muscle specimens using both restricted amounts of Duchenne/Becker's patient biopsy material [ 22 , 137 , 138 , 139 ] and especially a large variety of spontaneous or bio‐engineered animal models including dystrophic mice, pigs and dogs [ 21 , 23 , 99 , 127 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 ]. General listings of proteomic biomarkers of dystrophinopathy, covering both muscle tissue and biofluids such as serum, have been published in extensive reviews [ 11 , 12 , 13 , 14 , 15 , 24 , 56 ].…”

“…The table gives an overview of individual proteoforms identified by mass spectrometry, the various bioanalytical approaches taken for comparative analyses and the specific skeletal muscle tissues that were studied. The technical approaches for large‐scale protein separation included subcellular fractionation [ 54 , 156 ], membrane agglutination [ 99 ], isoelectric focussing [ 157 ], one‐dimensional gel electrophoresis [ 153 , 154 , 156 ], two‐dimensional gel electrophoresis [ 22 , 142 , 146 , 148 , 160 , 161 , 162 ] and reversed phase liquid chromatography [ 22 , 99 , 139 , 143 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 ], as well as chemical crosslinking [ 156 ]. Protein digestion protocols involved in‐gel, on‐membrane or in‐solution treatment with trypsin plus other types of proteases.…”

“…Labelling protocols encompassed differential fluorescent tagging prior to gel electrophoretic separation using difference gel electrophoresis (2D‐DIGE) [ 22 , 142 , 146 , 148 , 162 ], stable isotope labelling by amino acids in cell culture (SILAC) [ 147 ], isotope‐coded protein labelling (ICPL) [ 153 ] and tandem mass tagging (TMT) [ 145 , 157 ]. Multidimensional protein identification technology (MudPIT) [ 145 ], liquid chromatography tandem mass spectrometry (LC‐MS/MS) [ 22 , 99 , 139 , 143 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 ] and matrix assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐ToF) [ 142 , 160 , 161 , 162 ] were applied for routine protein identification. The identified changes in Ca 2+ ‐handling proteins using top‐down/gel‐based versus bottom‐up/gel‐free approaches were found to be mostly complementary and confirmatory in nature.…”

“…This would cause both reduced levels of Ca 2+ ‐ions being available for the passive release process during excitation–contraction coupling and altered patterns of Ca 2+ ‐dependent signalling in the cytosol [ 32 , 33 , 34 ]. In addition, the proteomic identification of lower abundances of SERCA‐type Ca 2+ ‐ATPases in dystrophic fibres suggests also impaired Ca 2+ ‐reuptake into the lumen of the sarcoplasmic reticulum during muscle relaxation [ 139 , 143 , 154 , 157 ]. Hence, systematic mass spectrometric surveys of dystrophic skeletal muscles have clearly established significant changes in key proteins involved in the regulation of the excitation–contraction–relaxation cycle.…”