Top-Down Proteomics of Skinned Human Muscle Fibers Reveals Proteoform-Resolved Fiber-to-Fiber Variability

Human skeletal muscle is composed of highly heterogeneous single muscle fibers (multinucleated single cells) that are commonly classified as fast or slow fiber types, yet proteoform-resolved characterization of individual human muscle fibers remains lacking. Herein, we establish a high sensitivity top-down proteomics method for the analysis of single human muscle fibers (hSMFs). Specifically, we have optimized the surfactant-free extraction protocol for analysis of chemically permeabilized ("skinned") hSMFs, a common preparation used to isolate the sarcomere prior to contractile measurements. This approach enables robust and reproducible proteoform-level coverage of key sarcomeric proteins from individual fibers using top-down LC-MS/MS. With this method, we identified extensive inter- and intra-donor fiber-to-fiber variability in isoform expression and proteoform abundance in hSMFs extracted from the heterogeneous vastus lateralis muscles. Together, these results demonstrate the capability of single-fiber top-down proteomics to resolve proteoform-level heterogeneity in human skeletal muscle and establish a methodological foundation for future studies towards elucidating skeletal muscle biology and understanding muscle-related diseases. Source data for this manuscript is available via the MassIVE repository at massive.ucsd.edu with identifier: MSV000100493.