Biochemical and proteomic insights into sarcoplasmic reticulum Ca ²⁺ -ATPase complexes in skeletal muscles

How Can Proteomics Help to Elucidate the Pathophysiological Crosstalk in Muscular Dystrophy and Associated Multi-System Dysfunction?

“…The isolation of distinct muscle fractions most frequently involves the microdissection of cellular structures or optimized tissue homogenization followed by differential centrifugation, density gradient ultracentrifugation and/or biochemical affinity isolation approaches [ 48 , 49 , 50 ]. Affinity purification can be carried out with pharmacological agents, immobilized lectins or suitable antibodies.…”

“…The main feature of the cellular pathogenesis of DMD is progressive myonecrosis, which is accompanied by chronic inflammation, reactive myofibrosis and an impaired regenerative capacity due to satellite cell dysfunction, as recently reviewed [ 258 ]. Skeletal muscles are characterized by the presence of extremely large proteins, i.e., giant proteins such as titin and nebulin [ 99 ], as well as many distinctly hydrophobic membrane proteins [ 50 ] and complex layers of mostly insoluble ECM components [ 259 ]. This makes proteomic studies of total tissue extracts technically challenging.…”

“…Figure 1 provides an overview of the major steps that are involved in routine proteomic analyses of proteins and their specific proteoforms. The most frequently employed methods in skeletal muscle proteomics have recently been reviewed, including top-down approaches, bottom-up techniques, comparative studies and membrane protein analyses [48][49][50]. The main procedures are briefly summarized in the below subsections, which outline the basic rationale of top-down versus bottom-up proteomics, protein separation, sample handling, the importance of optimized protein digestion, mass spectrometric techniques and data acquisition.…”

Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology

“…The production of precursor messenger RNA and alternative RNA splicing play a crucial part in the post-transcriptional regulation of gene expression. For example, the sarcoplasmic reticulum Ca 2+ -ATPase exists in the form of several protein isoforms with developmental and fiber type-specific expression patterns which derive through alternative RNA splicing from two genes [283] (Figure 9). Extensive PTMs are key mechanisms that produce distinct proteoforms, the functional protein units of cellular biological processes.…”

“…The two protein products are biochemically very different protein species; one is a typical transmembrane-spanning glycoprotein of the sarcolemma, beta-dystroglycan, and the other is an extracellular laminin receptor located in the basal lamina, alpha-dystroglycan (Figure 9). isoforms with developmental and fiber type-specific expression patterns which derive through alternative RNA splicing from two genes [283] (Figure 9). Extensive PTMs are key mechanisms that produce distinct proteoforms, the functional protein units of cellular biological processes.…”