Abstract
Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research.
Highlights
From the ‡Howard Hughes Medical Institute, §Senator Paul D
We present a novel biochemical workflow for the isolation, identification, and mass spectrometry-based quantification of protein complexes on the plasma membrane of mouse skeletal muscle, and report on several important discoveries related to muscular dystrophy using this workflow
These samples were subjected to lectin-affinity chromatography using wheat-germ agglutinin, and eluted with N-acetyl glucosamine
Summary
Animals—Animal care, ethical usage, and procedures were approved and performed in accordance with the standards set forth by the National Institutes of Health and the Animal Care Use and Review Committee at the University of Iowa. The beads were washed 3x in washing buffer (0.1% digitonin, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, proteinase inhibitors), and proteins were eluted by 1-hour incubation with 0.3 M N-acetyl-D-glucosamine in washing buffer, at 4 °C. The beads were washed 3x in washing buffer (0.1% digitonin, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, proteinase inhibitors), and proteins were eluted by 1-h incubation with 0.3 M N-acetyl-D-glucosamine in washing buffer, at 4 °C. WGA-enriched proteins were incubated with antibody beads, followed by washing 3ϫ (0.1% digitonin, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, proteinase inhibitors), and eluted in 0.1 M triethylamine, pH 11.5. The application of orthogonal protein fractionation methods (i.e. sucrose gradient density centrifugation, and strong-cation exchange peptide chromatography) coupled with the directed mass spectrometry workflow facilitated an in-depth proteomic comparison of skeletal muscle tissue between wildtype and muscular dystrophy mouse models. Statistical analyses were performed as described elsewhere [40]
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