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IsoTopics™ – July 2016

Determining Synthesis Rates of Individual Proteins in Zebrafish Danio rerio with Low Levels of a Stable Isotope Labelled Amino Acid

The zebrafish (Danio rerio) is an established model organism for studying cardiovascular disease. To better understand protein flux in the myocardium, Doherty MK et al. fed zebrafish low levels of L-Leu (d7), then measured its synthesis and turnover rates over an eight week labeling period. Bottom-up LC-MS/MS analysis of the heart tissue digests revealed rates of protein synthesis and incorporation for 661 proteins. These were predominantly intracellular, involved in metabolic processes and enriched in nine biochemical pathways (including glycolysis, ATP synthesis, and blood coagulation). This study marks an important step toward integrating genotype-phenotype data.


The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labeling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labeling regimens showing a good correlation of synthesis rates. 

Geary B, Magee K, Cash P, Young IS, Whitfield PD, Doherty MK.

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