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Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

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Pacific Northwest National Laboratory, Richland, Washington 99354, United States
*Phone: 509 371-6801. Fax: 509 376-9064. E-mail: [email protected]
Cite this: Anal. Chem. 2017, 89, 24, 13559–13566
Publication Date (Web):November 22, 2017
https://doi.org/10.1021/acs.analchem.7b03984
Copyright © 2017 American Chemical Society

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    Abstract

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    Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse–chase stable isotope labeling experiment. During the pulse, rats (n = 5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin and analyzed with liquid chromatography–tandem mass spectrometry (LC–MS/MS). MaxQuant was used to identify peptides and proteins and quantify heavy/light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3–103 days. For the ∼70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing turnover measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein biomarkers through better understanding of processes governing biomarker kinetics.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03984.

    • Proteins identified using global proteomic techniques (XLSX)

    • Turnover rates of proteins identified using global proteomic techniques (XLSX)

    • Proteins identified using activity-based protein profiling (XLSX)

    • Turnover rates of proteins identified using activity-based protein profiling (XLSX)

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    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

    Cited By

    This article is cited by 2 publications.

    1. Ethan G. Stoddard, Regan F. Volk, James P. Carson, Cecilia M. Ljungberg, Taylor A. Murphree, Jordan N. Smith, Natalie C. Sadler, Anil K. Shukla, Charles Ansong, Aaron T. Wright. Multifunctional Activity-Based Protein Profiling of the Developing Lung. Journal of Proteome Research 2018, 17 (8) , 2623-2634. https://doi.org/10.1021/acs.jproteome.8b00086
    2. Michael G. Friedrich. Spontaneous Breakdown of Long‐Lived Proteins in Aging and Their Implications in Disease. 2021, 97-125. https://doi.org/10.1002/9783527826759.ch4

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