Fig. 1.

Proteomic workflow, MS-based muscle fiber type assignment and phenotype. (A) MS-based proteomics workflow for the preparation and analysis of single muscle fibers from bed rest volunteers, whole lysates from astronauts, and samples for library generation. For details see main text and Materials and Methods. (B) Fiber-type assignment based on the % expression of four adult isoforms of MYH in 233 single muscle fibers. Fibers of each type were ranked by percentage purity. Both pure and mixed fibers are shown in the heatmap. The total number of pure and mixed fibers of each type in the dataset is indicated. Each vertical line represents a single fiber. Slow-1 fibers were defined as pure when expressing > 80% of MYH, 2A, and 2X when expressing > 70% of MYH2 and MYH1, respectively. The other fibers were defined as mixed-type. The adult isoform MYH4 (gray font) is only present in trace amount in human limb muscles. (C) Principal component analysis (PCA) of pure fibers (circles) and mixed-type fibers (dots), using only proteins expressed in all fibers. N = 233 pure and mixed-type single fibers, 178 proteins. (D) PCA loadings, with proteins driving the separation of slow-1 fibers in blue and of fast 2A and 2X in black. Proteins annotated as mitochondrial are significantly enriched in slow fibers (P = 1E-15). N = 233 single fibers, 178 proteins.