Deuterium labeling enables proteome-wide turnover kinetics analysis in cell culture

Protein turnover is a critical component of gene expression regulation and cellular homeostasis, yet methods for measuring turnover rates that are scalable and applicable to different models are still needed. We introduce an improved D₂O (heavy water) labeling strategy to investigate the landscape of protein turnover in cell culture, with accurate calibration of per-residue deuterium incorporation in multiple cell types. Applying this method, we mapped the proteome-wide turnover landscape of pluripotent and differentiating human induced pluripotent stem cells (hiPSCs). Our analysis highlights the role of APC/C (anaphase-promoting complex/cyclosome) and SPOP (speckle-type POZ protein) degrons in the fast turnover of cell-cycle-related and DNA-binding hiPSC proteins. Upon pluripotency exit, many short-lived hiPSC proteins are depleted, while RNA-binding and -splicing proteins become hyperdynamic. The ability to identify fast-turnover proteins also facilitates secretome profiling, as exemplified in hiPSC-cardiomyocyte and primary human cardiac fibroblast analysis. This method is broadly applicable to protein turnover studies in primary, pluripotent, and transformed cells.