Protocol to measure protein half-life in cell culture using heavy water

Protein turnover, comprising the continued synthesis and clearance of proteins, is required for protein homeostasis and cell survival. Here, we present a protocol to measure protein turnover half-life in cultured cells using D 2 O labeling. We describe steps to determine the extent of deuterium exchange in amino acids, perform dynamic labeling, and collect protein samples, followed by mass spectrometry and kinetic analysis. The protocol is suitable for measuring protein half-life under steady-state conditions and perturbations in multiple cell types and culture media. For complete details on the use and execution of this protocol, please refer to Alamillo et al. 1 • Steps for measuring cellular protein half-life with D 2 O labeling and mass spectrometry • Procedures for constructing and analyzing calibration standard cells • Instructions for analyzing labeling data to calculate protein half-life Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Protein turnover, comprising the continued synthesis and clearance of proteins, is required for protein homeostasis and cell survival. Here, we present a protocol to measure protein turnover half-life in cultured cells using D 2 O labeling. We describe steps to determine the extent of deuterium exchange in amino acids, perform dynamic labeling, and collect protein samples, followed by mass spectrometry and kinetic analysis. The protocol is suitable for measuring protein half-life under steady-state conditions and perturbations in multiple cell types and culture media.