Abstract
Metabolic labeling of living organisms with stable isotopes has become a powerful tool for global protein quantitation. The SILAC (stable isotope labeling with amino acids in cell culture) approach is based on the incorporation of nonradioactive-labeled isotopic forms of amino acids into cellular proteins. The effective SILAC labeling of immortalized cells and single-cell organisms (e.g., yeast and bacteria) was recently extended to more complex organisms, including worms, flies, and even rodents. The administration of a 13C6-lysine (heavy) containing diet for one mouse generation leads to a complete exchange of the natural (light) isotope 12C6-lysine. SILAC-labeled organisms are mainly used as a heavy “spike-in” standard into nonlabeled counterparts, and the combination with high-performance mass spectrometers allows for global proteomic screening. Here we used the fully labeled SILAC mice to identify proteins based on SILAC pairs from isolated cardiomyocytes, and we analyzed β-parvin-deficient hearts. Our approach confirmed the absence β-parvin and revealed simultaneously a clear up regulation of α-parvin in heart tissue. In this protocol, we describe the generation of a SILAC mouse colony and show two approaches to perform a proteome-wide analysis of heart tissue. Thus, the SILAC mouse spike-in approach is a readily available procedure and allows for a straightforward systematic analysis of disease models and knockout mice.
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Acknowledgments
This work was supported by the Max Planck Society, the Excellence Initiative “Cardiopulmonary System,” and the University of Giessen-Marburg Lung Center (UGMLC). In addition, we like to thank Silantes for the development of the Lys6 diet and Ingo Thievessen for providing the β-parvin knockout mice.
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Konzer, A., Ruhs, A., Braun, T., Krüger, M. (2013). Global Protein Quantification of Mouse Heart Tissue Based on the SILAC Mouse. In: Vivanco, F. (eds) Heart Proteomics. Methods in Molecular Biology, vol 1005. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-386-2_4
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DOI: https://doi.org/10.1007/978-1-62703-386-2_4
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-385-5
Online ISBN: 978-1-62703-386-2
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