RT Journal Article
SR Electronic
T1 Harmonizing labeling and analytical strategies to obtain protein turnover rates in intact adult animals
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.12.13.472439
DO 10.1101/2021.12.13.472439
A1 Dean E Hammond
A1 Deborah M Simpson
A1 Catarina Franco
A1 Marina Wright Muelas
A1 John Waters
A1 R W Ludwig
A1 Mark C Prescott
A1 Jane L Hurst
A1 Robert J Beynon
A1 Edward Lau
YR 2021
UL http://biorxiv.org/content/early/2021/12/23/2021.12.13.472439.abstract
AB 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.Competing Interest StatementThe authors have declared no competing interest.