Abstract
BackgroundSkeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. Each fiber has a specific molecular expression of myosin heavy chain molecules (MyHC). So far, MyHCs are currently the best marker proteins for characterization of individual fiber types, and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers.MethodsHerein, we describe a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited quantities of protein in single fibers, we developed a Proteomics high-throughput fiber typing (ProFiT) approach enabling profiling of MyHC in single fibers. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC, and phosphoproteomic analysis.ResultsOur fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of the Lys-6 incorporation rates in muscle fibers showed an increased turnover of slow fibers compared to fast fibers. In addition, labeling of mitochondrial respiratory chain complexes revealed a broad range of Lys-6 incorporation rates, depending on the localization of the subunits within distinct complexes.ConclusionOverall, the ProFiT approach provides a versatile tool to rapidly characterize muscle fibers and obtain fiber-specific proteomes for different muscle groups.
Highlights
Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions
Distinct protein patterns in intact skeletal muscle tissue and separated muscle fibers To provide an initial overview of the protein differences between skeletal muscle tissue and separated fibers, we extracted proteins from intact soleus skeletal muscles and a pool of ~ 50 isolated muscle fibers
To highlight the biological function of the proteins exclusively detected in term analysis of 300 proteins that were exclusively detected in isolated fibers showed that protein ubiquitination and helicase activity were overrepresented
Summary
Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. The identification of different myosin heavy chain isoforms (MyHC) represented a breakthrough in the molecular understanding of muscle fibers and has enabled a better understanding of the molecular heterogeneity of skeletal muscles [4, 5]. In addition to pure fibers expressing only one MyHC isoform, a number of mixed fibers—including type I/IIa, type IIa/IIx, and type IIb/IIx—contribute to the variability of muscle fibers in skeletal muscle tissues. Troponin, tropomyosin, and the calcium pump SERCA exist as slow and fast isoforms, which affect the performance and contractility of fiber types [5]. Due to their varied properties, each muscle fiber type responds differently to external stimuli, including exercise, starvation, or loss of neuronal innervation.
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