Spatial Proteomic Approach to Characterize Skeletal Muscle Myofibers

Abstract

Skeletal muscle myofibers have differential protein expression resulting in functionally distinct slow- and fast-twitch types. While certain protein classes are well-characterized, the depth of all proteins involved in this process is unknown. We utilized the Human Protein Atlas (HPA) and the HPASubC tool to classify mosaic expression patterns of staining across 49,600 unique tissue microarray (TMA) images using a visual proteomic approach. We identified 2164 proteins with potential mosaic expression, of which 1605 were categorized as “likely” or “real.” This list included both well-known fiber-type-specific and novel proteins. A comparison of the 1605 mosaic proteins with a mass spectrometry (MS)-derived proteomic dataset of single human muscle fibers led to the assignment of 111 proteins to fiber types. We additionally used a multiplexed immunohistochemistry approach, a multiplexed RNA-ISH approach, and STRING v11 to further assign or suggest fiber types of newly characterized mosaic proteins. This visual proteomic analysis of mature skeletal muscle myofibers greatly expands the known repertoire of twitch-type-specific proteins.

Spatial metabolomics reveals skeletal myofiber subtypes

“…However, our understanding has been limited by rudimentary biochemical techniques, e.g., fast/slow myosin ATPase immunostaining, or enzyme histochemistry for reduced form of nicotinamide adenine dinucleotide hydrogen (NADH) tetrazolium reductase, succinate dehydrogenase, and cytochrome c oxidase, which are nonquantitative and low-dimensional techniques where only a few enzymes can be analyzed at a time. A few studies have discussed the possibility of metabolic analysis of slow-and fast-twitch fibers, and some have studied single, isolated myofibers ex vivo (20)(21)(22)(23). However, there are still many technical obstacles to overcome, and none have performed metabolomic analysis of myofibers in vivo hitherto.…”

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

“…Subproteomic studies of skeletal muscle preparations were directed towards the surveying of proteins that are highly enriched in the sarcolemma [92], sarcoplasmic reticulum [175,304], mitochondrion-sarcoplasmic reticulum linker complexes [305], mitochondria [306][307][308] and the sarcosol [309], as well as the class of giant muscle proteins, such as dystrophin, nebulin, obscurin, plectin, the ryanodine receptor and the half-sarcomere spanning filamentous component titin [310]. Of note, the more recent application of single-cell proteomics (SCP) for studying fast versus slow myofibers [36] has revealed further molecular features of the extensive complexity of fiber type-specific protein expression patterns [33][34][35]261,277,[311][312][313].…”

“…The proteomic information summarizes the methodological approaches taken to establish the core protein components of skeletal muscles. The proteomic studies and reviews listed include (i) the initiation of the skeletal muscle proteome project [21,22], (ii) the systematic cataloguing of human skeletal muscles [20,23,26,27,37,53], (iii) proteomic surveys of fast versus slow human muscles [29,292], (iv) the analysis of human single myofibers [35,36,313], (v) the systematic cataloguing of animal skeletal muscles [25,28,288,290,[328][329][330], (vi) the differential proteomic surveys of fast versus slow mouse muscles [30][31][32], (vii) profiling of animal single myofibers [25,33,277,311,312], (viii) the mass spectrometric analysis of muscle spindles [295], (ix) the profiling of the neuromuscular junction [296,297], (x) proteomic profiling of subcellular fractions (sarcolemma, sarcoplasmic reticulum, mitochondria, sarcosol, giant muscle protein assemblies, contractile apparatus) [92,140,175,[304][305][306][308][309][310], (xi) the systematic cataloguing of the muscle secretome…”

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

“…Large-scale proteomic studies have especially focussed on the systematic profiling of normal human skeletal muscle specimens (vastus lateralis, various diagnostic biopsy specimens) [118][119][120][121] and the comparison of human fibre types (vastus lateralis, trapezius and deltoideus) [122][123][124][125], as well as the characterisation of wild type mouse skeletal muscle (gastrocnemius, leg muscle tissue, diaphragm) [21,[126][127][128], the comparison of rodent fibre types (gastrocnemius, soleus, tibialis anterior and extensor digitorum longus) [129][130][131][132][133] and single muscle fibre analyses of mouse muscles (soleus and extensor digitorum longus) [134][135][136].…”

“…Skeletal muscle proteomics has made enormous technical advances over the last two decades and has resulted in the mass spectrometric identification of over 10,000 muscle‐associated proteoforms [ 117 ]. Large‐scale proteomic studies have especially focussed on the systematic profiling of normal human skeletal muscle specimens ( vastus lateralis , various diagnostic biopsy specimens) [ 118 , 119 , 120 , 121 ] and the comparison of human fibre types ( vastus lateralis, trapezius and deltoideus ) [ 122 , 123 , 124 , 125 ], as well as the characterisation of wild type mouse skeletal muscle ( gastrocnemius , leg muscle tissue, diaphragm) [ 21 , 126 , 127 , 128 ], the comparison of rodent fibre types ( gastrocnemius, soleus, tibialis anterior and extensor digitorum longus ) [ 129 , 130 , 131 , 132 , 133 ] and single muscle fibre analyses of mouse muscles ( soleus and extensor digitorum longus ) [ 134 , 135 , 136 ]. These comprehensive cataloguing studies have clearly confirmed the presence of diverse protein constituents in the skeletal muscle proteome that are involved in the regulation of excitation–contraction coupling, the maintenance of triad structure and the mediation of spatial and temporal changes in Ca 2+ ‐related signalling events.…”