Figure 1.

Summary of experimental design. After 3 weeks on synthetic chow diet for acclimatization, young (4 month old) and old (26 month old) female mice had baseline echocardiography and were placed on a synthetic diet ad libitum (control group), 40% caloric restriction (progressively over 3 weeks), or ad libitum plus microencapsulated rapamycin 2.24 mg kg⁻¹day⁻¹. After 10 weeks, echocardiography was repeated and the mice were switched to the same synthetic diet but with ²H₃-leucine fully substituted for normal leucine. Tissue from 3 mice per treatment were harvested 3, 7, 12, and 17 days thereafter. Protein extraction was followed by LC-MS/MS. Topograph was applied to calculate the fraction of newly synthesized peptides, as well as the relative abundance of all isotopomers. The percentage newly synthesized peptides for each protein were plotted for the triplicate mice at each time point to derive the rate constant and half-lives based on first order kinetics. For abundance analysis, Topograph aligned the chromatograms to normalize the recovery times of the corresponding ions in each sample and obtain the areas under the curve for every peptide identified in any one sample. (see method S1 for further detail).