Duplexing metabolic deuterated water-labeled samples using dimethyl labeling to estimate protein turnover rates

Metabolic labeling with deuterated water (D₂O), combined with liquid chromatography coupled to mass spectrometry (LC-MS), is used to study turnover rates of individual proteins in vivo. Often, protein turnover rates from two (treatment and control) conditions are compared. The turnover rates are obtained from time course LC-MS experiments for each condition. Here, we explore dimethyl duplexing to directly compare label enrichment from two D₂O-labeled samples in a single LC-MS experiment. Light and heavy dimethyl channels carry two metabolically labeled samples. Protein turnover is modeled as a time course of the ratio of the monoisotopic relative abundance of a peptide in the heavy dimethyl channel to that in the light dimethyl channel. The turnover rates computed using the duplexed samples were close to those of non-duplexed samples. We illustrate the advantages of this method through the analysis of liver proteome dynamics in rapamycin-treated mice compared to control mice.