OBJECTIVES: In Inclusion Body Myositis (IBM), myofibers undergo structural and functional changes, including increased regeneration, atrophy, and fibrosis. The molecular mechanisms driving pathologically -associated fibers (PAF) remain poorly understood. METHODS: We developed a myofiber-level proteomic workflow to identify protein signatures of three PAF subtypes. Laser-capture microdissection mass spectrometry of immunolabeled cryosections was performed, complemented by immunofluorescence and electron microscopy validation. RESULTS: Regenerating fibers expressing embryonic myosin heavy chain showed greater molecular similarity to centrally nucleated fibers than to fibers adjacent to inflammation, which were enriched in aggregation-prone proteins. These distinct proteomic profiles revealed disruptions in protein homeostasis and proteasome composition, implicating impaired proteostasis in defective regeneration. In addition, alterations in HNRNPA1 subcellular localization across PAF subtypes suggest a potential role in driving protein aggregation and inflammation in IBM. CONCLUSIONS: This study underscores the value of spatial proteomics for dissecting localized pathological processes in muscle disease. It highlights the molecular heterogeneity of IBM myofibers and suggests that PAF subtype-specific mechanisms underlie impaired regeneration while pointing to potential drivers of IBM pathology. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-026-02720-7.