Thesis (Ph. D.)--University of Rochester. Department of Biology, 2017.
Living cells continuously degrade and resynthesize their constituent proteins. The maintenance of
protein homeostasis is fundamental to cell survival and function. Recent advances in mass
spectrometry, especially the development of stable isotope labeling with amino acids in cell culture
(SILAC), have enabled proteome-wide analyses of cellular protein turnover and elucidation of
protein homeostasis maintenance mechanisms. However, more efficient methods are still needed
for higher precision analysis of proteome dynamics. This work first clarified one overlooked issue
in the interpretation of dynamic SILAC experiments and indicated that in typical experiments
conducted in culture, the aminoacyl-tRNA precursor pool is near completely labeled in a few hours
and protein turnover is the limiting factor in establishing the labeling kinetics of most proteins.
Second, a methodology that combines metabolic isotopic labeling (SILAC) with isobaric tagging
(Tandem Mass Tags - TMT) was established for analysis of multiplexed samples. The described
methodology significantly reduces the cost and complexity of temporally-resolved dynamic
proteomic experiments and improves the precision of proteome-wide turnover data. By globally
quantifying the kinetics of protein clearance and synthesis, this approach provided important
insights into the regulation of the proteome as fibroblasts transit from a dividing to a quiescent state.
Our results indicated that, in quiescent cells, protein synthesis decreases, while protein degradation
increases by up-regulation of autophagy and lysosome biogenesis. Lastly, by measuring protein
degradation rates in wildtype and autophagy-deficient cells, we investigated the selectivity of
macroautophagy on a global scale. Together, this work developed a more efficient methodology
for measuring protein synthesis and turnover rates on a global scale and revealed an important
mechanism of protein homeostasis in quiescent fibroblasts.
