title: Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.
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.
authors.affiliation: Division of Health Research, University of the Highlands and Islands, Inverness, UK.
authors.forename: Bethany
authors.initials: B
authors.lastname: Geary
authors.affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
authors.forename: Kieran
authors.initials: K
authors.lastname: Magee
authors.affiliation: Division of Applied Medicine, University of Aberdeen, Aberdeen, UK.
authors.forename: Phillip
authors.initials: P
authors.lastname: Cash
authors.affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
authors.forename: Iain S
authors.initials: IS
authors.lastname: Young
authors.affiliation: Division of Health Research, University of the Highlands and Islands, Inverness, UK.
authors.forename: Phillip D
authors.initials: PD
authors.lastname: Whitfield
authors.affiliation: Division of Health Research, University of the Highlands and Islands, Inverness, UK.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Doherty
mesh_terms: D000818:Animals; D001692:Biological Transport; D005526:Food, Formulated; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D063990:Gene Ontology; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007930:Leucine / Q000378:metabolism*; D058977:Molecular Sequence Annotation; D009206:Myocardium / Q000378:metabolism*; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D020543:Proteome; D029961:Zebrafish Proteins; D007930:Leucine
keywords: Animal proteomics; Protein synthesis; Stable isotopes; Zebrafish