Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Hammond, Dean E, Simpson, Deborah M, Franco, Catarina, Muelas, Marina Wright, Waters, John, Ludwig, RW, Prescott, Mark C, Hurst, Jane L ORCID: 0000-0002-3728-9624, Beynon, Robert J ORCID: 0000-0003-0857-495X and Lau, Edward
(2022) Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals. MOLECULAR & CELLULAR PROTEOMICS, 21 (7). 100252-.

Access the full-text of this item by clicking on the Open Access link.


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 [<sup>13</sup>C<sub>6</sub>]lysine or [<sup>2</sup>H<sub>2</sub>]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.

Item Type: Article
Uncontrolled Keywords: Animals, Mice, Amino Acids, Lysine, Proteome, Isotope Labeling, Proteolysis
Divisions: Faculty of Health and Life Sciences
Faculty of Health and Life Sciences > Institute of Infection, Veterinary and Ecological Sciences
Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 10 Jun 2022 13:41
Last Modified: 18 Jan 2023 21:00
DOI: 10.1016/j.mcpro.2022.100252
Open Access URL: https://doi.org/10.1016/j.mcpro.2022.100252
Related URLs:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3156184