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

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.