Protein degradation is a critical regulatory mechanism for controlling protein levels. However, proteome-wide degradation measurements remain technically challenging, and even in the well-studied E. coli model, reliable measurements remain scarce.

Here, we quantify the degradation of ~3k E. coli proteins under 14 conditions by combining heavy isotope labeling with accurate multiplexed proteomics and find that E. coli recycles its cytoplasmic proteins when nitrogen-limited. Furthermore, we show that protein degradation rates do not scale with division rates. With knockout experiments, we identify substrates of the known ATP-dependent proteases but show that none is responsible for the cytoplasmic protein degradation in nitrogen starvation, suggesting that a major pathway in E. coli is still undiscovered. 

Thus, we introduce broadly-applicable technology for protein turnover measurements. We provide a rich resource for protein half-lives and protease substrates in E. coli that will allow researchers to complement genomics data to decipher the control of protein homeostasis.