Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals |
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Authors: | Dean E. Hammond Deborah M. Simpson Catarina Franco Marina Wright Muelas John Waters R.W. Ludwig Mark C. Prescott Jane L. Hurst Robert J. Beynon Edward Lau |
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Affiliation: | 1. Department of Biochemistry and System Biology, Institute of Systems and Integrative Biology, University of Liverpool, Liverpool, United Kingdom;2. Centre for Metabolomics Research, Institute of Systems and Integrative Biology, University of Liverpool, Liverpool, United Kingdom;3. Mammalian Behaviour & Evolution Group, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Leahurst Campus, Neston, United Kingdom;4. Department of Medicine/Cardiology, Consortium for Fibrosis Research and Translation, University of Colorado School of Medicine, Aurora, Colorado, USA |
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Abstract: | Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates. |
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Keywords: | protein turnover proteostasis heavy water protein degradation stable isotope labeling by amino acids AA" },{" #name" :" keyword" ," $" :{" id" :" kwrd0040" }," $$" :[{" #name" :" text" ," _" :" amino acid HW" },{" #name" :" keyword" ," $" :{" id" :" kwrd0050" }," $$" :[{" #name" :" text" ," _" :" heavy water MS" },{" #name" :" keyword" ," $" :{" id" :" kwrd0060" }," $$" :[{" #name" :" text" ," _" :" mass spectrometry MUP" },{" #name" :" keyword" ," $" :{" id" :" kwrd0070" }," $$" :[{" #name" :" text" ," _" :" major urinary protein RIA" },{" #name" :" keyword" ," $" :{" id" :" kwrd0080" }," $$" :[{" #name" :" text" ," _" :" relative isotope abundance precursor RIA plateau |
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