Abstract
Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. All data are available via ProteomeXchange with the identifier PXD025359. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach, identified a discriminative peptide panel, and confirmed our panel using a public data set.
Highlights
We used this approach for the precise excision of skeletal muscle fiber types out of complex muscle tissue using laser microdissection (LMD) followed by data-independent acquisition (DIA) mass spectrometry (MS) to uncover differences in four murine skeletal muscle fiber type proteomes
Our applied analysis workflow has the following advantages: (i) the protocol requires very little sample material, which makes it highly applicable for small samples, such as those obtained with skeletal muscle biopsies; (ii) our method is not dependent on postanalytical assignment of fiber types and enables ad priori distinction of the four fiber types by immunohistochemical staining and selective enrichment by laser microdissection
Both fiber types are referred to as fast, the literature states that type IIa fibers mainly sustain their energetic needs using oxidative pathways and, by containing high numbers of mitochondria, whereas type IIx fibers rely on glycolysis to produce ATP rapidly [5,10,68]
Summary
Fiber types are classically subdivided into slow type I and fast type
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