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

Abstract

Voluntary striated muscles are characterized by a highly complex and dynamic proteome that efficiently adapts to changed physiological demands or alters considerably during pathophysiological dysfunction. The skeletal muscle proteome has been extensively studied in relation to myogenesis, fiber type specification, muscle transitions, the effects of physical exercise, disuse atrophy, neuromuscular disorders, muscle co-morbidities and sarcopenia of old age. Since muscle tissue accounts for approximately 40% of body mass in humans, alterations in the skeletal muscle proteome have considerable influence on whole-body physiology. This review outlines the main bioanalytical avenues taken in the proteomic characterization of skeletal muscle tissues, including top-down proteomics focusing on the characterization of intact proteoforms and their post-translational modifications, bottom-up proteomics, which is a peptide-centric method concerned with the large-scale detection of proteins in complex mixtures, and subproteomics that examines the protein composition of distinct subcellular fractions. Mass spectrometric studies over the last two decades have decisively improved our general cell biological understanding of protein diversity and the heterogeneous composition of individual myofibers in skeletal muscles. This detailed proteomic knowledge can now be integrated with findings from other omics-type methodologies to establish a systems biological view of skeletal muscle function.

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

“…Enrichment strategies can be used to isolate and enrich sub-populations of peptides based on specific chemical properties [ 117 , 118 ]. A description of the analysis pipelines for investigating PTMs specific to skeletal muscle cell biology has recently been collated [ 48 ]. Phosphoproteomics has become one of the most active research areas in proteomic studies with phosphopeptide enrichment being a critical step in the analysis [ 119 , 120 ].…”

“…The proteomic profiling of skeletal muscles is concerned with both the detailed intra-proteomic analysis of the heterogenous cell types that are present within the neuromuscular system and inter-proteomic surveys focusing on the crosstalk between skeletal muscles and other organs via the circulatory system. Based on the dynamic nature of proteoforms as the basic units of the proteome [ 12 , 13 , 14 , 15 ], skeletal muscle proteomics attempts to increase our biochemical and pathophysiological knowledge of protein changes in health and disease [ 18 , 19 , 20 , 48 ]. However, since skeletal muscle tissues are heterogeneous in their cellular composition, this biological fact is reflected by the diverse constitution of the accessible muscle proteome [ 185 ].…”

“…Skeletal muscles contain both tissues that are difficult to homogenize and comprise a large amount of sarcomeric proteins, membrane-associated proteins and relatively insoluble proteins of the ECM, making the proteomic analysis of total extracts a difficult task. Proteome-wide effects on skeletal muscles are routinely studied using cultured muscle cells, human biopsy specimens and muscles derived from animal models [ 48 ]. The collection of mass spectrometrically identified and muscle-associated proteins consists of over 10,000 individual species.…”

“…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.…”

“…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.…”