High-Throughput Acoustic FFPE Proteomics Reveals ATP5IF1-Associated Mitochondrial Alterations in Acral Melanoma

Formalin-fixed, paraffin-embedded (FFPE) archives underpin dermatopathology and translational oncology, enabling clinically annotated melanoma cohorts, but cohort-scale proteomics remains limited by labor and variability in upstream processing. We established a plate-scale, acoustic FFPE proteomics workflow and integrated it with AI-assisted digital pathology to support composition-aware molecular profiling from routine sections. The optimized pipeline reduces handling steps, is designed to improve reproducibility, and supports rapid parallel processing in a 96-well format. Deep data-independent acquisition MS of 40 primary melanomas spanning acral lentiginous, lentigo maligna, superficial spreading, and nodular subtypes quantified more than 8200 protein groups and a mean of 5200 proteins per tumor. Proteome profiles resolved melanoma subtypes and defined an acral lentiginous melanoma program enriched for translation/biogenesis and extracellular matrix/adhesion processes with relative depletion of lipid and fatty-acid metabolism and peroxisomal pathways. Supervised feature selection highlighted tenascin, periostin, eukaryotic initiation factor 4A-I, and ADP-ribosylation factor 4 and uncovered selective depletion of ATPase inhibitor, mitochondrial (ATP5IF1), a regulator of mitochondrial ATP synthase, in acral lentiginous melanoma. ATP5IF1 depletion persisted after adjustment for mitochondrial proxies and QuPath-derived tumor content, and coincided with higher glycolysis relative to Complex V. This pathology-integrated, high-throughput FFPE proteomics framework enables scalable retrospective discovery and suggests subtype-specific metabolic alterations consistent with mitochondrial remodeling in an underrepresented melanoma subtype.