SUMMARY White adipocytes function as the major energy reservoir in humans by storing substantial amounts of triglycerides and their dysfunction is associated with metabolic disorders. However, the mechanisms underlying cellular specialization during adipogenesis remain unknown. Here, using a high-sensitivity-high throughput workflow, we generated a spatiotemporal proteomic atlas of human adipogenesis encompassing information for ~8.000 proteins. Our systematic approach gives insights into cellular remodeling and the spatial reorganization of metabolic pathways to optimize cells for lipid accumulation and highlights the coordinated regulation of protein localization and abundance during adipogenesis. More specifically, we identified a compartment-specific regulation of protein levels and localization changes of metabolic enzymes to reprogram branched chain amino acid and one-carbon metabolism to provide building blocks and reduction equivalents for lipid synthesis. Additionally, we identified C19orf12 as a differentiation induced and adipocyte-specific lipid droplet (LD) protein, which interacts with the translocase of the outer membrane (TOM) complex of LD associated mitochondria and regulates adipocyte lipid storage. Overall, our study provides a comprehensive resource for understanding human adipogenesis and for future discoveries in the field. KEY POINTS Human adipogenesis induces distinct temporal changes in protein abundance 20% of all detected proteins change organellar localization during adipogenesis BCAA and one-carbon metabolism enzyme levels and localizations are coordinately regulated to promote adipogenesis C19orf12 is an adipocyte specific LD protein and regulator of lipid storage

A Spatiotemporal Proteomic Map of Human Adipogenesis