Proteomics Moves From Expression to Turnover

Update and Future Perspective

Mary K Doherty; Phillip D Whitfield

Disclosures

Expert Rev Proteomics. 2011;8(3):325-334. 

In This Article

Proteome Turnover in Animals

Determining protein synthesis and degradation rates in cell culture is relatively uncomplicated in terms of experimental design. The ability to control and manipulate the RIA of the precursor pool means that the downstream interpretation of the mass spectra is routine, with the correct bioinformatic support. Moving to intact complex organisms requires careful consideration of label choice, introduction of label to the subject and recycling of unlabeled amino acid from pre-existing tissue. The label must be of sufficient abundance in the proteome of the organism and, if possible, should be administered via the diet to minimize stress. When designing the diet, issues of nutrition (i.e., sufficient vitamins and minerals) and palatability must be addressed. A fully synthetic diet may not be palatable and there are issues relating to bioavailability.

To address the viability of this approach in intact animals, Doherty et al.[34] built upon the principles of mass isotopomer distribution analysis[35–37] and fed chickens a semi-synthetic diet, where 50% of the valine had been replaced with either the unlabeled isotope or the 2H8- labeled amino acid. The birds were maintained on the light variant of the diet for 5 days to ensure palatability and then switched to the diet containing the labeled valine for a further 5 days. Birds were sampled at regular time intervals over the labeling period and the soluble proteins from the pectoralis muscle were analyzed. Identification of the proteins and labeled peptide pairs by MS was straightforward. However, as it was not possible to supply 100% of the valine in the deuterated form and owing to dilution of the label by recycled proteins from pre-existing tissues, it was necessary to empirically determine the extent of heavy label incorporation. This calculation of RIA was subsequently used to deconvolute the contribution of pre-existing and newly synthesized protein to the total protein pool. This has been described comprehensively in a previous review by Doherty and Beynon.[3]

The production of fully labeled SILAC mice has been an important advance in this area and could subsequently be used to determine protein synthesis and degradation rates.[38] In this work, a specific diet was developed, which permitted the complete labeling of a F2 generation of mice. Either 13C6- or 12C6-lysine was mixed into a customized lysine-free mouse diet to levels that fulfilled the nutritional requirements of the mice. It was found that there were no growth or developmental impairments associated with this diet over at least four generations. During the labeling period, blood was sampled on a weekly basis for 4 weeks. Label incorporation was monitored in a cohort of proteins showing variance in incorporation rates. Labeling efficiency was also measured in other tissues (heart, liver and gut epithelium) at the 4-week time point. As might be expected, label incorporation rates differed between the different tissues. However, as discussed previously, it is difficult to obtain complete labeling in a single generation; therefore, the labeled mice were bred for a number of generations with almost 93% incorporation detected in the F2 generation. In a more recent study, this work was extended to the study of proteome dynamics in the mouse.[39] Wild-type, inbred mice were fed a diet of algae, which had been ubiquitously labeled with 15N. This was achieved by growing Spirulina platensis in a bioreactor with Na15NO3 as the sole nitrogen source. The mice were subsequently fed the labeled algae for 32 days, with samples culled at certain time points during the labeling period. Proteins were analyzed from the brain, liver and blood, with turnover rates spanning four orders of magnitude. It was observed that brain proteins, in general, had a lower rate of turnover than those from liver and blood. Labeling of other model organisms, including Drosophila melanogaster and Caenorhabditis elegans[40] has also been achieved, although this has been for quantification rather than determination of protein turnover.[41]

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