Proteomics. 2016 May;16(9):1398-406.

Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

Geary B1, Magee K2, Cash P3, Young IS2, Whitfield PD1, Doherty MK1.
1 Division of Health Research, University of the Highlands and Islands, Inverness, UK.
2 Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
3 Division of Applied Medicine, University of Aberdeen, Aberdeen, UK.

 

Abstract

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 labelling 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 labelling regimens showing a good correlation of synthesis rates.

Animal proteomics; Protein synthesis; Stable isotopes; Zebrafish

PMID:26929125; DOI:10.1002/pmic.201500357

 

Supplement:

Human disease research can be constrained by the type and scale of experiments that are feasible for most researchers. The symptoms of many diseases in humans may take a considerable period of time to manifest, which can limit the availability of suitable patients covering the complete trajectory of the disease pathology. In addition, the complex genetic background of human populations means that larger and better-controlled subject groups are required. Critically, many experimental manipulations are not allowed to be performed on human patients due to ethical, legal and practical considerations.

 

The zebrafish (Danio rerio) has become an important model organism in biomedical research and is increasingly being employed to investigate the molecular basis of human diseases. The zebrafish, a member of the cyprinid family, originates from rivers in India, and is a common pet fish found in most aquariums. The zebrafish has a number key advantages. Zebrafish thrive and reproduce very readily in the laboratory environment. The adults are small in size, have a high fecundity, reproduce very readily and have a short generation interval allowing the rapid production of large colonies of fish. Moreover, the translucent body of zebrafish embryos facilitates non-intrusive visualisation of organs.  The zebrafish also shares a high genetic similarity to humans with approximately 70% of all human genes found in zebrafish (Howe et al 2013).

 

The zebrafish heart is widely studied and is the first organ to develop in the fish, with a functioning, beating heart by 22 hours post-fertilisation. Both human and zebrafish hearts have common functions and characteristics including the regulation of rhythm, the presence of pacemaker activity and the movement of blood flow directed by valves. The zebrafish heart also has an interesting capacity to regenerate, which has led to considerable interest in understanding the mechanisms of this process in relation to heart function and disease.

 

Proteomic technologies can be used to probe the functional link between expressed genes and phenotypic outcomes in disease processes. However, unlike the genome, which is considered the blueprint of an organism, the proteome is not a predetermined, static entity but in constant flux. Even in a position of apparent steady state, where the size of a protein pool remains constant, the protein complement is changing with new proteins being synthesized and older proteins being degraded and recycled.   Conventional comparative proteomic approaches do not address the intricate dynamics of the proteome but simply provide a ‘snap-shot’ of the proteome at a given time under defined conditions. As such, it provides little insight into the mechanism creating differences between biological states. To bridge this gap, it is not enough to measure the steady state levels of proteins rather protein turnover, the net result of protein synthesis and degradation, needs to be considered.

 

We have previously developed strategies to determine the absolute rates of protein synthesis and degradation in complex organisms (Doherty et al, 2005). The methodology involves administering a stable isotope labelled amino acid via the diet and monitoring its incorporation into proteins over time with mass spectrometry. This powerful approach enables the turnover of hundreds of individual proteins from a given tissue to be determined in a single experiment providing a dynamic picture of the proteome. In this study we successfully adapted these technologies to the zebrafish with a specific focus on cardiac muscle. Importantly, this was achieved through the formulation of a diet containing low-levels of [2H7]-L-leucine a considerable cost in these types of experiments. This is a key departure from previous studies and will facilitate a wider adoption of this experimental approach.

 

The translation of this technology to zebrafish has potentially huge implications. Dysfunction of protein synthesis and degradation pathways has been implicated in a number of disease states including cardiovascular disease, cancer and neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease. There already exist a number of zebrafish models that have mutations directly relevant to these conditions and experiments can be designed using non-mutant zebrafish to mimic the pathophysiological processes. The ability to define proteomic dynamics in these zebrafish models therefore offers new avenues of research to investigate the molecular mechanisms of human diseases and provide a more comprehensive view of the cellular control systems involved in complex pathologies.

 

 

Figure 1: Zebrafish are an important model organism for biomedical research. In this paper we focussed on the zebrafish heart, which has many features common to humans. The zebrafish heart also has the ability to regenerate following injury, allowing investigations into tissue remodelling and repair.

 

 

Figure 2: Zebrafish were fed a diet containing either 30% or 50% of its leucine as a stable isotope labelled form. Samples were taken over an eight week period and the proteome analysed. By determining the relative amounts of heavy and light label incorporated into each protein, it was possible to determine the absolute rate of protein synthesis of each protein identified. The rates obtained from both the 30% and 50% labelled diet showed good correlation, indicating that the use of a lower level of the labelled amino acid is a viable option.

 

References:

Doherty MK, Whitehead C, McCormack H, Gaskell SJ, Beynon RJ. Proteome dynamics in complex organisms: Using stable isotopes to monitor individual protein turnover rates. Proteomics 2005; 5: 522-533

Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 2013; 496: 498-503

 

 

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