Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. This methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. Our data indicate that fibroblasts selectively increase degradation rates of long-lived proteins as they transition from a proliferating to a quiescent state. The selective degradation of long-lived proteins occurs by the concurrent activation of lysosomal biogenesis and upregulation of macroautophagy.
Sina Ghaemmaghami is an assistant professor of biology at the University of Rochester. His research focuses on understanding the mechanisms of cellular protein folding and degradation with a special focus on neurodegenerative disorders. He has authored more than 30 publications in journals such as Science, Nature, and PNAS. His laboratory has developed a number of commonly used proteomic methodologies for global analyses of cellular protein homeostasis.