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

Publication Details

• Journal: Cell Rep Methods
• Volume: 5 (7 )
• Pages: 101104
• Published: Jul 2025
• Type: Journal Article

Abstract

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.

Links & Resources

Related Research Projects