{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T12:24:06Z","timestamp":1792499046771,"version":"4.5.0"},"reference-count":49,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2025,5,5]],"date-time":"2025-05-05T00:00:00Z","timestamp":1746403200000},"content-version":"vor","delay-in-days":4,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["physoc.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The Journal of Physiology"],"published-print":{"date-parts":[[2025,5]]},"abstract":"<jats:sec><jats:title>Abstract<\/jats:title><jats:p>Skeletal muscle is a complex syncytial arrangement of an array of cell types and, in the case of muscle\u2010specific cells (myofibres), subtypes. There exists extensive heterogeneity in skeletal muscle functional behaviour and molecular landscape at the cell composition, myofibre subtype and intra\u2010myofibre subtype level. This heterogeneity highlights limitations in currently applied methodological approaches, which has stagnated our understanding of fundamental skeletal muscle biology in both healthy and myopathic contexts. Here we developed a novel approach that combines a fluorescence\u2010based assay for the biophysical examination of the sarcomeric protein, myosin, coupled with same\u2010myofibre high\u2010sensitivity proteome profiling, termed single myofibre protein function\u2010omics (SMPFO). Applying this approach as proof\u2010of\u2010principle we identify the integrated relationship between myofibre functionality and the underlying proteomic landscape that guides divergent, but physiologically important, behaviour in myofibre subtypes in healthy human skeletal muscle. By applying SMPFO to two forms of human nemaline myopathy (<jats:italic>ACTA1<\/jats:italic> and <jats:italic>TNNT1<\/jats:italic> mutations), we reveal significant reduction in the divergence of myofibre subtypes across both biophysical and proteomic behaviour. Collectively we demonstrate preliminary findings of SMPFO to support its use to study skeletal muscle with greater specificity, accuracy and resolution than currently applied methods, facilitating that advancement in understanding of skeletal muscle tissue in both healthy and diseased states.\n<jats:boxed-text content-type=\"graphic\" position=\"anchor\"><jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" mimetype=\"image\/png\" position=\"anchor\" specific-use=\"enlarged-web-image\" xlink:href=\"graphic\/tjp16724-gra-0001-m.png\"><jats:alt-text>image<\/jats:alt-text><\/jats:graphic><\/jats:boxed-text><\/jats:p><\/jats:sec><jats:sec><jats:title>Key points<\/jats:title><jats:p><jats:list list-type=\"bullet\">\n<jats:list-item><jats:p>Skeletal muscle is a complex tissue made up of an array of cell and sub\u2010cell types, with the resident muscle cell \u2013 myofibre \u2013 critical for contractile function.<\/jats:p><\/jats:list-item>\n<jats:list-item><jats:p>Although single myofibre studies have advanced, existing methods lack the precision for simultaneous multidata analysis, hindering developments in our understanding of skeletal muscle.<\/jats:p><\/jats:list-item>\n<jats:list-item><jats:p>We introduce single myofibre protein function\u2010omics (SMPFO), a method enabling functional analysis of sarcomeric myosin alongside global protein abundance within the same myofibre.<\/jats:p><\/jats:list-item>\n<jats:list-item><jats:p>In healthy myofibres SMyoMFO reveals extensive biochemical diversity in myosin heads, correlating with the abundance of metabolic and sarcomeric proteins, including subtype\u2010specific patterns in sarcoglycan delta (SGCD).<\/jats:p><\/jats:list-item>\n<jats:list-item><jats:p>In contrast SMyoMFO uniquely reveals a reduction in diversity of myosin function and the myofibre proteome in two forms of nemaline myopathy, highlighting disease\u2010associated alterations.<\/jats:p><\/jats:list-item>\n<jats:list-item><jats:p>This innovative approach provides a robust framework for investigating myofibre regulation and dysfunction in skeletal muscle biology.<\/jats:p><\/jats:list-item>\n<\/jats:list><\/jats:p><\/jats:sec>","DOI":"10.1113\/jp288363","type":"journal-article","created":{"date-parts":[[2025,5,5]],"date-time":"2025-05-05T07:42:02Z","timestamp":1746430922000},"page":"3033-3048","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Integrated single\u2010cell functional\u2010proteomic profiling reveals a shift in myofibre specificity in human nemaline myopathy: A proof\u2010of\u2010principle study"],"prefix":"10.1113","volume":"603","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2491-3747","authenticated-orcid":false,"given":"Robert A. 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