Protein Turnover in Aging and Longevity

Abstract

Progressive loss of proteostasis is a hallmark of aging that is marked by declines in various components of proteostasis machinery, including: autophagy, ubiquitin-mediated degradation, protein synthesis, and others. While declines in proteostasis have historically been observed as changes in these processes, or as bulk changes in the proteome, recent advances in proteomic methodologies have enabled the comprehensive measurement of turnover directly at the level of individual proteins in vivo. These methods, which utilize a combination of stable-isotope labeling, mass spectrometry, and specialized software analysis, have now been applied to various studies of aging and longevity. Here we review the role of proteostasis in aging and longevity, with a focus on the proteomic methods available to conduct protein turnover in aging models and the insights these studies have provided thus far.

Keywords: aging; mass spectrometry; protein turnover; proteostasis; stable isotope labeling.

Figure 1

General workflow for measurement of in vivo protein turnover rates in rodents using mass spectrometry. A) The use of heavy‐labeled amino acids generally requires a synthetic diet of a similar composition to regular chow, and it is important to acclimate animals to the non‐labeled synthetic diet for a few weeks prior to the start of the experiment. Mouse treatments, if used, are usually administered prior to supplementation of heavy label. For heavy‐water labeling, an initial bolus of labeled water is injected at the start of the labeling period, followed by supplementation of a lower percentage of deuterium in the drinking water. Otherwise, label is supplemented in the chow during this period. Tissues from all treatment groups are then collected at several time points, usually on the order of days to weeks, and processed for mass spectrometry analysis. B) For comprehensive survey of turnover, samples are usually analyzed by mass spectrometry using data‐dependent acquisition. An analysis of peptide isotopomer peaks is then conducted using specialized software (e.g. Topograph) to determine the enrichment of label in the precursor pool and the percentage of each protein that is newly synthesized. For each protein, a regression of the fraction that is newly synthesized is then performed to determine its rate of turnover.