Reconstruction of the Last Eukaryotic Common Ancestor (LECA) protein interactome through integration of >26,000 mass spectrometry experiments across 31 species reveals 109,466 conserved protein interactions among 3,193 ancient gene families, which successfully predicts novel gene-disease associations for approximately one-third of tested Mendelian disorders and elucidates disease mechanisms including ciliary dysfunction in EFHC2-associated renal failure, ATP6V1A in osteopetrosis, and GLG1 in short-rib thoracic dysplasia.

All eukaryotes share a single-celled ancestor from ∼1.5–1.8 billion years ago, the Last Eukaryotic Common Ancestor (LECA). Roughly half of gene families found in modern eukaryotes were already present in LECA, forming molecular systems that continue to influence genetic diseases and traits today. To investigate these systems, we compared genes across 156 organisms to define a core set of protein-coding gene families likely present in LECA, with a quarter remaining uncharacterized. Integrating >26,000 mass spectrometry proteomics analyses from 31 species, we inferred higher-order complexes among these ancient proteins. This reconstructed interactome reveals both established and novel assemblies, offering a biochemical snapshot of LECA’s organization. Finally, by exploring these ancient protein interactions, we found new human gene-disease associations for bone density and congenital birth defects, illustrating the value of ancestral protein networks for modern functional genetics.